Conjugates And Conjugating Reagents Comprising A Linker That Includes At Least Two (-CH2-CH2-O-) Units In A Ring

ABSTRACT

A conjugate comprising a protein or peptide conjugated to a therapeutic, diagnostic or labelling agent via a linker, characterised in that the linker includes at least two ˜(CH 2 —CH 2 —O—)˜ units within a ring, said ring being attached via a single tethering atom within the ring to the rest of the linker, or said ring being attached via two or more tethering atoms within the ring to the rest of the linker at a single point.

FIELD OF INVENTION

This invention relates to novel conjugates and novel conjugatingreagents.

BACKGROUND OF THE INVENTION

Much research has been devoted in recent years to the conjugation of awide variety of payloads, for example therapeutic, diagnostic andlabelling agents, to peptides and proteins for a wide range ofapplications. The protein or peptide itself may have therapeuticproperties, and/or it may be a binding protein.

Peptides and proteins have potential use as therapeutic agents, andconjugation is one way of improving their properties. For example, watersoluble, synthetic polymers, particularly polyalkylene glycols, arewidely used to conjugate therapeutically active peptides or proteins.These therapeutic conjugates have been shown to alter pharmacokineticsfavourably by prolonging circulation time and decreasing clearancerates, decreasing systemic toxicity, and in several cases, displayingincreased clinical efficacy. The process of covalently conjugatingpolyethylene glycol, PEG, to proteins is commonly known as “PEGylation”.The PEG chain may carry a payload, for example a therapeutic, diagnosticor labelling agent.

Binding proteins, particularly antibodies or antibody fragments, arefrequently conjugated. The specificity of binding proteins for specificmarkers on the surface of target cells and molecules has led to theirextensive use either as therapeutic or diagnostic agents in their ownright or as carriers for payloads which may include therapeutic,diagnostic or labelling agents. Such proteins conjugated to labels andreporter groups such as fluorophores, radioisotopes and enzymes find usein labelling and imaging applications, while conjugation to drugs suchas cytotoxic agents and chemotherapy drugs to produce antibody-drugconjugates (ADCs) allows targeted delivery of such agents to specifictissues or structures, for example particular cell types or growthfactors, minimising the impact on normal, healthy tissue andsignificantly reducing the side effects associated with chemotherapytreatments. Such conjugates have extensive potential therapeuticapplications in several disease areas, particularly in cancer.Conjugates containing binding proteins frequently contain PEG.

Many methods of conjugating proteins and peptides have been reported inthe literature. Probably the most commonly used process involves the useof conjugating reagents based on maleimides or succinimides. Suchreagents are described in many publications, for example WO 2004/060965and WO 2013/090590. An alternative approach which leads to morehomogeneous products is described by Liberatore et al, Bioconj. Chem1990, 1, 36-50, and del Rosario et al, Bioconj. Chem. 1990, 1, 51-59,which describe the use of reagents which may be used to cross-linkacross the disulfide bonds in proteins, including antibodies. WO2005/007197 describes a process for the conjugation of polymers toproteins, using novel conjugating reagents having the ability toconjugate with both sulfur atoms derived from a disulfide bond in aprotein to give novel thioether conjugates, while WO 2009/047500describes the use of the same conjugating reagents to bond topolyhistidine tags attached to the protein. WO 2010/100430 describesreagents capable of forming a single carbon bridge across the disulfidebond in a protein. Other documents relating to the conjugation ofproteins include WO 2014/064423, WO 2013/190292, WO 2013/190272 and EP2260873.

WO 2014/064424 describes specific ADCs in which the drug is a maytansineand the antibody is bonded by cross-linking across a disulfide bond. WO2014/064423 describes specific ADCs in which the drug is an auristatinand the antibody is bonded by cross-linking across a disulfide bond. Thelinkers illustrated in the Examples of these documents contain a PEGportion as part of the backbone of the linker, in which one end of thelinear PEG chain is attached via a further portion of the linker to thedrug, while the other end of the PEG chain is attached via a furtherportion of the linker to the antibody. This is a common structuralpattern for ADCs. WO 2015/057699 and US 2016/0000933 describealternative structures in which a linear PEG chain is attached to thebackbone of the linker.

WO 2009/152440 describes a small molecule conjugate compound comprisinga targeting ligand; a therapeutic agent and/or an imaging agent; and alinker connecting the ligand to the therapeutic agent and/or the imagingagent. A very long list of possible linkers is included (para. 0060),and this includes crown ether (for chelating with metal). WO 2007/055700is concerned with lanthanide chelates derived from diazacrown ethershaving two ethyliminodiacetic acid side chains. Again, the crown etheris present because of its chelating ability.

Over recent years, the importance of the linker which links a payload tothe protein or peptide in a conjugate, has become apparent. Often, thekey decision to be taken is whether it is desired to have a cleavablelinker, i.e. a linker which, on administration of the conjugate,degrades to release the free payload, or a non-cleavable linker. Anotherkey decision is whether or not to include PEG in the linker. Subject tothese considerations, in principle, any linker may be used. In practice,however, changes in structure of the linker may lead to differences inthe properties either of the conjugating reagent or of the resultingconjugate.

One problem frequently found is that conjugates may be less storagestable than desired. This is particularly true when a cleavable linkeris used, when it is desired that the conjugate should have a longshelf-life before administration but should then rapidly cleave onapplication, but it can be true for any linker. There is a need toincrease the storage stability of conjugates. In addition, improvedstability in vivo is desirable, as this can lead to increased biologicalactivity. There is also a need to increase biological activity. Finally,there is a need to improve conjugation efficiency when making theconjugates, to provide highly homogeneous products, and to minimiseaggregation during the conjugation process. We have now found that for aparticular class of conjugate, the use of PEG-containing linkers of aparticular structure, gives advantageous properties. Specifically,conjugates with high stability and/or potency may be obtained.

SUMMARY OF THE INVENTION

The invention provides a conjugate comprising a protein or peptideconjugated to a therapeutic, diagnostic or labelling agent via a linker,characterised in that the linker includes at least two ˜(CH₂—CH₂—O—)˜units within a ring, said ring being attached via a single tetheringatom within the ring to the rest of the linker, or said ring beingattached via two or more tethering atoms within the ring to the rest ofthe linker at a single point.

The invention also provides a conjugating reagent comprising afunctional group capable of reacting with a protein or peptide, whichreagent also comprises a therapeutic, diagnostic or labelling agent anda linker which includes at least two ˜(CH₂—CH₂—O—)˜ units within a ring,said ring being attached via a single tethering atom within the ring tothe rest of the linker, or said ring being attached via two or moretethering atoms within the ring to the rest of the linker at a singlepoint.

The invention also provides a process for the preparation of a conjugateaccording to the invention, which comprises reacting a protein orpeptide with a conjugating reagent according to the invention, said ringbeing attached via a single tethering atom within the ring to the restof the linker, or said ring being attached via two or more tetheringatoms within the ring to the rest of the linker at a single point.

DETAILED DESCRIPTION OF THE INVENTION

The reagent of the invention may be represented schematically by theformula:

in which D represents the therapeutic, diagnostic or labelling agent, Frepresents a functional grouping capable of bonding to a protein orpeptide, and

represents a ring which includes at least two ethylene glycol,˜(CH₂—CH₂—O—)˜, units. The functional grouping F is capable of reactingwith a protein or peptide as explained in more detail below.

The conjugate of the invention may be represented schematically by theformula:

in which D represents the therapeutic, diagnostic or labelling agent, F′represents the protein or peptide bonded to the remainder of theconjugate via a protein or peptide bonding portion of the linker, and

represents a ring which includes at least two ethylene glycol,˜(CH₂—CH₂—O—)˜, units.

The Polyethylene Glycol Ring

Throughout this specification, “polyethylene glycol ring” should beunderstood to mean a ring which includes at least two ethylene glycol,˜(CH₂—CH₂—O—)˜, units. The ring may include two or more separate˜(CH₂—CH₂—O—)˜ units, or it may include one or more units of the formula˜(CH₂—CH₂—O—)_(x)˜ in which x is a number of at least 2. The ring maycontain one or more additional atoms to complete the cyclic structure.Additional atoms may for example be nitrogen, carbon, oxygen, sulfur,silicon and/or phosphorus atoms.

It is a feature of the present invention that the ring is pendant to therest of the linker rather than forming part of the backbone of thelinker, i.e. it is attached to the rest of the linker but capable whenin solution of moving flexibly relative to the rest of the linker. It isbelieved that, in contrast to the situation where the ring forms part ofthe backbone of the linker, as described in WO 2007/055700, this enablesthe conjugate, when in solution, to adopt a more flexible conformationthan when the ring is rigidly held within the backbone of the linker,giving improved properties. Both of WO 2007/055700 and WO 2009/152440include a ring in the linker in order to chelate a metal ion, which is aquite different objective to that of the present invention.

To this end, the ring may be attached via a single tethering atom withinthe ring to the rest of the linker at a single point, or it may beattached at two or more points. Alternatively, the ring may be attachedvia two or more tethering atoms within the ring to the rest of thelinker at a single point. Tethering atoms may for example be nitrogen,carbon, phosphorus or silicon atoms, especially nitrogen and/or carbonatoms, and the atoms present at the point of attachment to the rest ofthe linker may for example be nitrogen or carbon atoms.

The following are schematic drawings of possible forms of attachment ofthe ring to the rest of the linker in conjugates or reagents of theinvention, T representing a tethering atom in the ring, and PEGrepresenting at least two ˜(CH₂—CH₂—O—)˜ units:

Specific examples of suitable rings include the following, where thesymbol ˜ indicates a point of incorporation of the ring into the linker:

Preferably the ring is attached to the rest of the linker at a singlepoint, and most preferably the ring is attached via a single tetheringatom in the ring to the rest of the linker at a single point.

The ring may for example consist of ˜(CH₂—CH₂—O—)_(x)˜ units in which xis at least 2, preferably from 2 to 20. Alternatively, the ring maycontain ˜(CH₂—CH₂—O—)_(x)˜ units in which x is at least 2, preferablyfrom 2 to 50, especially from 2 to 20, but may also include one or moreadditional atoms as mentioned above, or may be derivatised in some otherway.

Conjugates and reagents may be readily synthesised from crown ethers.Crown ethers are cyclic oligomers of ethylene glycol, and many differentcrown ethers are known, some of which consist entirely of ethyleneglycol units, and some of which contain additional atoms within thering. For example, aza-crown ethers contain a nitrogen atom, whilediaza-crown ethers contain two nitrogen atoms. Many crown ethers arecommercially available, and these provide convenient starting points forsynthesis of the conjugates and reagents according to the invention.Crown ethers carrying functional groups through which they may bereacted with other compounds are known, for example crown etherscarrying carboxy, hydroxy, amino, or aldehyde groups are known, as arecrown ethers fused to a benzene ring optionally carrying a functionalgroup such as a carboxy, hydroxy, amino, isocyanate, nitro or aldehydegroup.

Crown ethers are known to chelate cations, and perfluoro crown ethershave been described within U.S. Pat. No. 4,838,274 for use in MM.Therefore the conjugates of the invention may be used in applicationswithin imaging techniques such as MM or PET.

Typical crown ethers which can be incorporated into the conjugates andreagents according to the invention include the structures shown below.

These may be attached to the backbone of the linker of the conjugatesand reagents of the invention by reaction through atoms, especiallynitrogen atoms, present within the ring, or via groups, for examplehydroxy, amino, carboxy, aldehyde, isocyanate or nitro groups, presenton a side-chain. Typical linkages are as shown below:

In addition to rings derived from crown ethers, rings derived fromcryptands may be used in the present invention. Such rings are describedfor example in US 2014/0072900, and include the following:

The conjugates and reagents of the invention may contain one ringincluding at least two ˜(CH₂—CH₂—O—)˜ units, or they may contain two ormore such rings. The ring may be monocyclic, or it may be bi- ormulti-cyclic. Two or more rings may be attached to the backbone of thelinker, or they may be attached to each other, thus:

It will be understood that many different sizes and structures of ringsare possible. The important feature of the invention is that a PEG chainforms part of a cyclic structure: this chain is not a linear PEG chainwhich forms part of the backbone of the linker, neither is it a pendantPEG chain which is tethered at one end to the linker but which has afree untethered end. Rather, it is a pendant PEG-containing cyclicstructure which does not form part of the backbone of the linker.

In one preferred embodiment, all of the PEG in the conjugate or reagentaccording to the invention is present within one or more rings. Inanother embodiment, PEG may also be present elsewhere in the linker,specifically in the backbone of the linker or in a group linking thering to the backbone of the linker, and this is discussed in more detailbelow.

The total number of ˜(CH₂—CH₂—O—)˜ units present in the conjugates andreagents of the invention will of course depend on the intendedapplication. For some applications, high molecular weight PEGs may beused, for example the number average molecular weight may be up toaround 75,000, for example up to 50,000, 40,000 or 30,000 g/mole. Forexample, the number average molecular weight may be in the range of from500 g/mole to around 75,000. However, smaller PEG portions may bepreferred for some applications.

As with the total quantity of PEG present in the conjugates or reagentsof the invention, the number of ˜(CH₂—CH₂—O—)˜ units present in the ringwill depend on the intended application. For example the cyclic PEGportion may have a molecular weight up to 3,000 g/mole. However, cyclicgroups containing as few as 2 ethylene glycol units, for example from 2to 50 ethylene glycol units, are useful for some applications, and arepresent as a cyclic PEG group in one preferred embodiment of theinvention. PEG-containing rings with 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19 or 20 repeat units, or 24, 36, 40 or 48repeat units, may for example be used.

The Payload

The conjugates and reagents of the invention carry a payload which is atherapeutic, diagnostic or labelling agent. A single molecule of atherapeutic, diagnostic or labelling agent may be present, or two ormore molecules may be present. The inclusion of one or more drugmolecules, for example a cytotoxic agent or a toxin, is preferred.Auristatins, maytansinoids and duocarmycins are typical cytotoxic drugs.It is often preferred that drug conjugates, particularly antibody drugconjugates, should contain multiple copies of the drug. Labelling agents(which should be understood to include imaging agents) may for exampleinclude a radionuclide, a fluorescent agent (for example an aminederivatised fluorescent probe such as5-dimethylaminonaphthalene-1-(N-(2-aminoethyl))sulfonamide-dansylethylenediamine, Oregon Green® 488 cadaverine (catalogue number 0-10465,Molecular Probes), dansyl cadaverine,N-(2-aminoethyl)-4-amino-3,6-disulfo-1,8-naphthalimide, dipotassium salt(lucifer yellow ethylenediamine), or rhodamine B ethylenediamine(catalogue number L 2424, Molecular Probes), or a thiol derivatisedfluorescent probe for example BODIPY® FL L-cystine (catalogue numberB-20340, Molecular Probes). Biotin may also be used.

Preferably the payload is a therapeutic agent, especially one of thosementioned above.

The Protein

For convenience in this section and elsewhere, “protein” should beunderstood to include “protein and peptide” except where the contextrequires otherwise.

Suitable proteins which may be present in the conjugates of theinvention include for example peptides, polypeptides, antibodies,antibody fragments, enzymes, cytokines, chemokines, receptors, bloodfactors, peptide hormones, toxin, transcription proteins, or multimericproteins.

Enzymes include carbohydrate-specific enzymes, proteolytic enzymes andthe like, for example the oxidoreductases, transferases, hydrolases,lyases, isomerases and ligases disclosed by U.S. Pat. No. 4,179,337.Specific enzymes of interest include asparaginase, arginase, adenosinedeaminase, superoxide dismutase, catalase, chymotrypsin, lipase,uricase, bilirubin oxidase, glucose oxidase, glucuronidase,galactosidase, glucocerbrosidase, glucuronidase, and glutaminase.

Blood proteins include albumin, transferrin, Factor VII, Factor VIII orFactor IX, von Willebrand factor, insulin, ACTH, glucagen, somatostatin,somatotropins, thymosin, parathyroid hormone, pigmentary hormones,somatomedins, erythropoietin, luteinizing hormone, hypothalamicreleasing factors, antidiuretic hormones, prolactin, interleukins,interferons, for example IFN-α or IFN-β, colony stimulating factors,hemoglobin, cytokines, antibodies, antibody fragments,chorionicgonadotropin, follicle-stimulating hormone, thyroid stimulatinghormone and tissue plasminogen activator.

Other proteins of interest are allergen proteins disclosed by Dreborg etal Crit. Rev. Therap. Drug Carrier Syst. (1990) 6 315-365 as havingreduced allergenicity when conjugated with a polymer such aspoly(alkylene oxide) and consequently are suitable for use as toleranceinducers. Among the allergens disclosed are Ragweed antigen E, honeybeevenom, mite allergen and the like.

Glycopolypeptides such as immunoglobulins, ovalbumin, lipase,glucocerebrosidase, lectins, tissue plasminogen activator andglycosylated interleukins, interferons and colony stimulating factorsare of interest, as are immunoglobulins such as IgG, IgE, IgM, IgA, IgDand fragments thereof.

Of particular interest are receptor and ligand binding proteins andantibodies and antibody fragments which are used in clinical medicinefor diagnostic and therapeutic purposes. Antibody-drug conjugates, basedon an antibody or an antibody fragment, especially where the drug is acytotoxic drug, for example an auristatin, maytansinoid or duocarmycin,are an especially preferred embodiment of the invention. Except wherethe context requires otherwise, any reference in this Specification to aconjugate of the invention should be understood to include a specificreference to an antibody drug conjugate.

An example of an antibody which may be useful in the conjugates of theinvention is an anti-CD30 antibody, for example a chimeric monoclonalantibody cAC10, e.g. brentuximab. Brentuximab is the antibody portion ofbrentuximab vedotin (INN), trade name Adcetris®. Brentuximab vedotin isdefined by the WHO as follows:

Immunoglobulin G1-kappa auristatin E conjugate, anti-[Homo sapiensTNFRSF8 (tumor necrosis factor receptor superfamily member 8, KI-1,CD30)], chimeric monoclonal antibody conjugated to auristatin E; gamma1heavy chain (1-446) [Mus musculus VH (IGHV1-84*02-(IGHD)-IGHJ3*01)[8.8.10] (1-117) -Homo sapiens IGHG1*01 CH₃ K130>del (118-446)],(220-218′)-disulfide (if not conjugated) with kappa light chain(1′-218′) [Mus musculus V-KAPPA (IGKV3-4*01-IGKJ1*01) [10.3.9] (1′-111′)-Homo sapiens IGKC*01 (112′-218′)]; (226-226″)-disulfide dimer;conjugated, on an average of 3 to 5 cysteinyl, to monomethylauristatin E(MMAE), via a maleimidecaproyl-valyl-citrullinyl-p-aminobenzylcarbamate(mc-valcit-PABC) linker

For the vedotin part, please refer to the document “INN forpharmaceutical substances: Names for radicals, groups and others”*.

Heavy chain QIQLQQSGPE VVKPGASVKI SCKASGYTFT DYYITWVKQK PGQGLEWIGW 50IYPGSGNTKY NEKFKGKATL TVDTSSSTAF MQLSSLTSED TAVYFCANYG 100NYWFAYWGQG TQVTVSAAST KGPSVFPLAP SSKSTSGGTA ALGCLVKDYF 150PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTQTYIC 200NVNHKPSNTK VDKKVEPKSC DKTHTCPPCP APELLGGPSV FLFPPKPKDT 250LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY 300RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT 350LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS 400DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG 446 Light chainDIVLTQSPAS LAVSLGQRAT ISCKASQSVD FDGDSYMNWY QQKPGQPPKV 50LIYAASNLES GIPARFSGSG SGTDFTLNIH PVEEEDAATY YCQQSNEDPW 100TFGGGTKLEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKV 150QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV 200THQGLSSPVT KSFNRGEC 218

Disulfide Bridges Location

Intra-H 22-96 144-200 261-321 367-425 22″-96″ 144″-200″ 261″-321″367″-425″ Intra-L 23′-92′ 138′-198′ 23′′′-92′′′ 138′′′-198′′′ Inter-H-L* 220-218′ 220″-218″ Inter-H-H*  226-226″ 229-229″ *Two or three of theinter-chain disulfide bridges are not present, the antibody beingconjugated to an average of 3 to 5 drug linkers each via a thioetherbond.

N-Glycosylation Sites

297, 297″

(WHO Drug Information, Vo. 25, No. 1, 2011; International NonproprietaryNames for Pharmaceutical Substances (INN), Recommended InternationalNonproprietary Names, List 65)

The protein may be derivatised or functionalised if desired. Inparticular, prior to conjugation, the protein, for example a nativeprotein, may have been reacted with various blocking groups to protectsensitive groups thereon; or it may have been previously conjugated withone or more polymers or other molecules. It may contain a polyhistidinetag, which during the conjugation reaction can be targeted by theconjugating reagent.

Conjugating Reagents and Processes

Many conjugating reagents which can be used to conjugate a payload to aprotein are known, and the novel conjugating reagents of the inventiondiffer from these known reagents in the nature of the linker theycontain. Such reagents contain at least one functional group F capableof reacting with a protein or peptide. For example, the conjugatingreagent may comprise a functional group capable of reacting with atleast one electrophile or, especially, nucleophile, present in theprotein, the functional group being attached to the payload via thelinker.

Any type of known conjugation reaction may be used to form theconjugates of the invention. For example, the reaction may be carriedout using the known methods of thiol bonding, amine conjugation, orclick chemistry. For example, the reagent may contain a maleimide group,an N-hydroxysuccinimide group, a click-chemistry group, for example anazide or alkyne group, an amine group, a carboxyl group, a carbonylgroup, or an active ester group. Other possible approaches include theuse of proteins that have been recombinantly engineered with an aminoacid specifically for conjugation such as engineered cysteines ornon-natural amino acids, and enzymatic conjugation through a specificenzymatic reaction such as with transglutaminase. The reaction site onthe protein may be either nucleophilic or electrophilic in nature.Common protein conjugation sites are at lysine or cysteine amino acidresidues or carbohydrate moieties. Alternatively, conjugation may occurat a polyhistidine tag which has been attached to a binding protein.

A conjugating reagent according to the invention is advantageouslycapable of reacting with a nucleophile in a protein and hence becomingchemically bonded thereto. As such the conjugating reagent typicallyincludes at least one leaving group which is lost on reaction with anucleophile. The conjugating reagent may, for example, include two ormore leaving groups. Preferably the conjugating reagent according to theinvention is capable of reacting with two nucleophiles. Advantageously,the conjugating reagent according to the invention comprises at leasttwo leaving groups. If two or more leaving groups are present, these maybe the same or different. Alternatively, a conjugating reagent maycontain a single group which is chemically equivalent to two leavinggroups and which single group is capable of reacting with twonucleophiles.

Nucleophilic groups include sulfur atoms and amine groups, andnucleophilic groups in proteins are for example provided by cysteine,lysine or histidine residues. In one preferred embodiment of theinvention, a nucleophilic group is a sulfur atom present in a cysteineresidue present in the protein. Such structures may be obtained byreduction of a disulfide bond present in the protein. In anotherembodiment, a nucleophilic group may be an imidazole group present in ahistidine residue present in a polyhistidine tag attached to theprotein.

One group of reagents is based on the bis-halo- or bis-thio-maleimidesand derivatives thereof as described in Smith et al., J. Am. Chem. Soc.,2010, 132, 1960-1965, and Schumacher et al., Bioconj. Chem., 2011, 22,132-136. These reagents contain the functional grouping:

in which each L is a leaving group. The nitrogen atom of the maleimidering is connected to the linker.

Similarly, maleimides containing a single leaving group L:

may be used. Again, the nitrogen atom of the maleimide ring is connectedto the linker.

Also, maleimides lacking a leaving group:

may be used. Again, the nitrogen atom of the maleimide ring is connectedto the linker.

Another group of reagents are those described within WO201173391Anexample of these reagents comprises the functional grouping:

in which each L is a leaving group.

In an especially preferred embodiment of the invention, the conjugatingreagent contains the functional grouping F:

in which W represents an electron-withdrawing group, for example a ketogroup, an ester group —O—CO—, or a sulfone group —SO₂—; each of A and Bindependently represents a C₁₋₅alkylene or alkenylene chain; and eithereach L independently represents a leaving group, or both Ls togetherrepresent a leaving group. When reagents containing such groups reactwith proteins, a first leaving group L is lost to form in situ aconjugating reagent containing a functional grouping of formula:

in which m is 0 to 4, which reacts with a first nucleophile. The secondleaving group L is then lost, and reaction with a second nucleophileoccurs. As an alternative to using a reagent containing the functionalgrouping I as starting material, reagents containing the functionalgrouping II may be used, as the functional groupings I and II arechemical equivalents of each other.

These conjugating reagents of the invention are of the general typedisclosed in WO 2005/007197 and WO 2010/100430. Such reagents may forexample be used to target two sulfur atoms obtained by reduction of adisulfide bond in a protein, or imidazole groups present in histidineresidues present in a polyhistidine tag attached to a protein. It hasbeen found that the incorporation of a cyclic PEG group according to thepresent invention into reagents of this type gives particularly goodresults, with conjugation reactions occurring efficiently to producestable conjugates with a high degree of homogeneity.

A leaving group L may for example be —SP, —OP, —SO₂P, —OSO₂P, —N⁺PR²R³,halogen, or —OØ, in which P represents a hydrogen atom or an alkyl(preferably C₁₋₆alkyl), aryl (preferably phenyl), or alkyl-aryl(preferably C₁₋₆alkyl-phenyl) group, or is a group which includes aportion —(CH₂CH₂O)_(n)— in which n is a number of two or more, and eachof R² and R³ independently represents a hydrogen atom, a C₁₋₄ alkylgroup, or a group P, and Ø represents a substituted aryl, especiallyphenyl, group, containing at least one substituent, for example —CN,CF₃, —NO₂, —CO₂R^(a), —COH, —CH₂OH, —COR^(a), —OR^(a), —OCOR^(a),—OCO₂R^(a), —SR^(a), —SOR^(a), —SO₂R^(a), —NHCOR^(a), —NR^(a)COR^(a),—NHCO₂R^(a), —NR^(a)CO₂R^(a), —NO, —NHOH, —NR^(a) OH, —CH═N—NR^(a)COR^(a), —N⁺R^(a) ₃, —, halogen, especially chlorine or, especially,fluorine, —C≡CR^(a), and —CH═CR^(a) ₂, in which each R^(a) independentlyrepresents a hydrogen atom or an alkyl (preferably C₁₋₆alkyl), aryl(preferably phenyl), or alkyl-aryl (preferably C₁₋₆alkyl-phenyl) group.The presence of electron withdrawing substituents is preferred.

Conjugating reagents in which P represents a group which includes aportion —(CH₂CH₂O)_(n)— in which n is a number of two or more are thesubject of our copending application GB 1418186, published as WO2016/059377. This application discloses the following:

-   -   “The leaving group may for example include —(CH₂CH₂O)_(n)—R¹        where R¹ is a capping group. A very wide range of capping groups        may be used. R¹ may for example be a hydrogen atom, an alkyl        group, especially a C₁₋₄alkyl group, particularly a methyl        group, or an optionally substituted aryl group, for example an        optionally substituted phenyl group, for example a tolyl group.        Alternatively, the capping group may include a functional group        such as a carboxyl group or an amine group. Such capping groups        may for example have the formula —CH₂CH₂CO₂H or —CH₂CH₂NH₂, and        may be prepared by functionalising the terminal unit of a        —(CH₂CH₂O)_(n)— chain. Alternatively, rather than being        terminated by a capping group, the —(CH₂CH₂O)_(n)— group may        have two points of attachment within the conjugating reagent        such that chemically the equivalent of two leaving groups are        present, capable of reacting with two nucleophiles.    -   The —(CH₂CH₂O)_(n)— portion of the leaving group is based on        PEG, polyethylene glycol. The PEG may be straight-chain or        branched, and it may be derivatised or functionalised in any        way. n is a number of 2 or more, for example 2, 3, 4, 5, 6, 7,        8, 9 or 10. For example, n may be from 5 to 9. Alternatively, n        may be a number of 10 or more. There is no particular upper        limit for n. n may for example be 150 or less, for example 120        or less, for example 100 or less. For example n may be from 2 to        150, for example from 7 to 150, for example from 7 to 120. The        PEG portion —(CH₂CH₂O)_(n)— of a leaving group may for example        have a molecular weight of from 1 to 5 kDa; it may for example        be 1 kDa, 2 kDa, 3 kDa, 4 kDa or 5 kDa. A leaving group may if        desired contain two or more portions —(CH₂CH₂O)_(n)— separated        by one or more spacers.    -   A leaving group in a conjugating reagent according to the        invention is suitably of the formula —SP, —OP, —SO₂P, —OSO₂P,        —N⁺PR²R³, in which P is a group which includes a portion        —(CH₂CH₂O)_(n)— and each of R² and R³ independently represents a        hydrogen atom, a C₁₋₄ alkyl group, or a group P. Preferably each        of R² and R³ represents a C₁₋₄alkyl group, especially a methyl        group, or, especially, a hydrogen atom. Alternatively, the        conjugating reagent may include a group of formula —S—P—S—;        —O—P—O—; —SO₂—P—SO₂—; —OSO₂—P—OSO₂—; and —N⁺R²R³—P—N⁺R²R³—.        Specific groups of this type include —S—(CH₂CH₂O)_(n)—S—,        —O—(CH₂CH₂O)_(n)—O—; —SO₂—(CH₂CH₂O)_(n)—SO₂—;        —OSO₂—(CH₂CH₂O)_(n)—OSO₂—; or —N⁺R²R³—(CH₂CH₂O)_(n)—N⁺R²R³—.        They can also include groups of the type:

-   -   where the —(CH₂CH₂O)_(n)— group is carried by any suitable        linking group, for example an alkyl group. These divalent groups        are chemically equivalent to two leaving groups capable of        reacting with two nucleophiles.”

An especially preferred leaving group L present in a novel conjugatingreagent according to the present invention is —SP or —SO₂P, especially—SO₂P. Within this group, one preferred embodiment is where P representsa phenyl or, especially, a tolyl group. Another preferred embodiment iswhere P represents a group which includes a portion —(CH₂CH₂O)_(n)—,especially one in which n has one of the values mentioned above,especially 7. An especially preferred leaving group L is—SO₂—(CH₂CH₂O)_(n)—H/Me, especially —SO₂—(CH₂CH₂O)₇—H/Me. Throughoutthis Specification, any reference to a leaving group L should beunderstood to include a specific reference to these preferred groups,especially —SO₂—(CH₂CH₂O)_(n)—H/Me, and more especially—SO₂—(CH₂CH₂O)₇—H/Me.

Preferably W represents a keto group. Preferably each of A and Brepresents —CH₂—.

Reagents of the formula I and II above form conjugates which include thegrouping F′:

in which W′ represents an electron withdrawing group or a group obtainedby reduction of an electron withdrawing group, and Pr represents aprotein or peptide bonded to A and B via nucleophiles Nu. The immediateproduct of the conjugation process (as described in more detail below)is a conjugate which contains an electron-withdrawing group W. However,the conjugation process is reversible under suitable conditions. Thismay be desirable for some applications, for example where rapid releaseof the protein is required, but for other applications, rapid release ofthe protein may be undesirable. It may therefore be desirable tostabilise the conjugates by reduction of the electron-withdrawing moietyto give a moiety which prevents release of the protein. Accordingly, theconjugation process may comprise an additional optional step of reducingthe electron withdrawing group in the conjugate. The use of aborohydride, for example sodium borohydride, sodium cyanoborohydride,potassium borohydride or sodium triacetoxyborohydride, as reducing agentis particularly preferred.

Other reducing agents which may be used include for example tin (II)chloride, alkoxides such as aluminium alkoxide, and lithium aluminiumhydride.

Thus, for example, a moiety W containing a keto group may be reduced toa moiety containing a CH(OH) group; an ether group CH.OR^(a) may beobtained by the reaction of a hydroxy group with an etherifying agent;an ester group CH.O.C(O)R^(a) may be obtained by the reaction of ahydroxy group with an acylating agent; an amine group CH.NH₂, CH.NHR^(a)or CH.NR^(a) ₂ may be prepared from a ketone by reductive amination; oran amide CH.NHC(O)R^(a) or CH.N(C(O)R^(a))₂ may be formed by acylationof an amine; in which R^(a) represents a hydrogen atom or an alkyl(preferably C₁₋₆ alkyl), aryl (preferably phenyl), or alkyl-aryl(preferably C₁₋₆alkyl-phenyl) group. A sulfone may be reduced to asulfoxide, sulfide or thiol ether.

Preferably the groupings F′ and F have the formula:

especially

In the above formulae, preferred leaving groups are as described above.Preferably each Nu is a sulfur atom.

Another preferred group of conjugating reagents contains the functionalgrouping:

˜W—CR⁴R^(4′)—CR⁴.L.L′  (IV)

in which W has the meaning and the preferred meanings given above, andeither

each R⁴ represents a hydrogen atom or a C₁₋₄ alkyl group, R^(4′)represents a hydrogen atom, and either each L independently represents aleaving group, or both Ls together represent a leaving group; or

each R⁴ represents a hydrogen atom or a C₁₋₄ alkyl group, L represents aleaving group, and R^(4′) and L′ together represent a bond.

Another group of conjugating reagents includes the functional grouping:

˜W—(CH═CH)_(p)—(CH₂)₂-L  (V) or

˜W—(CH═CH)_(p)—CH═CH₂  (VI)

in which W has the meaning and preferred meanings given above and prepresents 0 or an integer of from 1 to 4, preferably 0. An especiallypreferred reagent of this type includes the functional grouping:

˜NH—CO—Ar—CO—(CH₂)₂-L  (Va) or

˜NH—CO—Ar—CO—CH═CH₂  (VIa)

in which Ar represents an optionally substituted aryl, especiallyphenyl, group.

In all cases, preferred meanings for leaving groups L and L′ are asmentioned above.

Conjugating reagents according to the invention may contain more thanone functional grouping for reaction with a protein. For example, areagent may contain a functional grouping, preferably of formula I orII, at one end of the molecule, and one or more additional functionalgroupings, elsewhere in the molecule. Such structures are described infor example Belcheva et al, J. Biomater. Sci Polymer Edn. 9(3), 207-226and are useful in the synthesis of conjugates containing multipleproteins.

The novel conjugating reagents of the present invention may be preparedby methods analogous to known methods. Specific synthesis reactions areillustrated in the Examples which follow.

Conjugating reagents according to the invention may be reacted with aprotein or peptide to form a conjugate according to the invention, andsuch a reaction forms a further aspect of the invention. Thus, aconjugating reagent including a suitable functional grouping, especiallythe functional grouping I or II, is reacted with a protein or peptide,especially an antibody or antibody fragment, to form a conjugate,especially one including the grouping III.

A key feature of using conjugating reagents of the formulae I or II isthat an α-methylene leaving group and a double bond are cross-conjugatedwith an electron withdrawing function that serves as a Michaelactivating moiety. If the leaving group is prone to elimination in thecross-functional reagent rather than to direct displacement and theelectron-withdrawing group is a suitable activating moiety for theMichael reaction then sequential intramolecular bis-alkylation can occurby consecutive Michael and retro Michael reactions. In reagentscontaining the functional grouping I, a leaving group serves to mask alatent conjugated double bond that is not exposed until after the firstalkylation has occurred to give a reagent including the functionalgrouping II and bis-alkylation results from sequential and interactiveMichael and retro-Michael reactions. The cross-functional alkylatingagents may contain multiple bonds conjugated to the double bond orbetween the leaving group and the electron withdrawing group.

Where bonding to the protein is via two sulfur atoms derived from adisulfide bond in the protein, the process may be carried out byreducing the disulfide bond following which the reduced product reactswith the reagent according to the invention. Preferably the disulfidebond is reduced and any excess reducing agent is removed, for example bybuffer exchange, before the conjugating reagent is introduced. Thedisulfide bond can be reduced, for example, with dithiothreitol,mercaptoethanol, or tris-carboxyethylphosphine using conventionalmethods.

Conjugation reactions may be carried out under similar conditions toknown conjugation processes, including the conditions disclosed in WO2005/007197, WO 2009/047500, WO 2014/064423, WO 2014/064424 and WO2015/057699. The process may for example be carried out in a solvent orsolvent mixture in which all reactants are soluble. For example, theprotein may be allowed to react directly with the polymer conjugatingreagent in an aqueous reaction medium. This reaction medium may also bebuffered, depending on the pH requirements of the nucleophile. Theoptimum pH for the reaction will generally be at least 4.5, typicallybetween about 5.0 and about 8.5, preferably about 6.0 to 7.5. Theoptimal reaction conditions will of course depend upon the specificreactants employed.

Reaction temperatures between 3-40° C. are generally suitable when usingan aqueous reaction medium. Reactions conducted in organic media (forexample THF, ethyl acetate, acetone, DMSO, DMF, MeCN) are typicallyconducted at temperatures up to ambient. In one preferred embodiment,the reaction is carried out in aqueous buffer which may contain aproportion of organic solvent, for example up to 20% by volume oforganic solvent, typically from 5 to 20% by volume of organic solvent.

The protein can be effectively conjugated using a stoichiometricequivalent or a slight excess of conjugating reagent. However, it isalso possible to conduct the conjugation reaction with an excessstoichiometry of conjugating reagent, and this may be desirable for someproteins. The excess reagent can easily be removed, for example by ionexchange chromatography or HPLC, during subsequent purification of theconjugate.

Of course, it is possible for more than one conjugating reagent to beconjugated to a protein, where the protein contains sufficient suitableattachment points. For example, in a protein which contains twodifferent disulfide bonds, or in a protein which contains one disulfidebond and also carries a polyhistidine tag, it is possible to conjugatetwo molecules of reagent per molecule of protein, and such conjugatesform part of the present invention.

The Linker

The conjugates and reagents of the present invention contain a linkerwhich connects the therapeutic, diagnostic or labelling agent to theprotein or peptide in the conjugates of the invention or to thefunctional grouping in conjugating reagents of the invention. Thebackbone of the linker is a continuous chain of atoms which runs fromthe therapeutic, diagnostic or labelling agent at one end to the proteinor peptide at the other end. This linker must include one or more cyclicPEG portions, i.e. rings including at least two —(CH₂—CH₂—O)— units, asdescribed above. It may also contain any other desired groups,particularly any of the conventional groups commonly found in thisfield.

Subsection (i). In one embodiment, the linker between the payload andthe grouping of formula F′/F, and particularly that portion of thelinker immediately adjacent the grouping of formula F′/F, may include analkylene group (preferably a C₁₋₁₀ alkylene group), or anoptionally-substituted aryl or heteroaryl group, any of which may beterminated or interrupted by one or more oxygen atoms, sulfur atoms,—NR^(a) groups (in which R^(a) represents a hydrogen atom or an alkyl(preferably C₁₋₆alkyl), aryl (preferably phenyl), or alkyl-aryl(preferably C₁₋₆alkyl-phenyl) group), keto groups, —O—CO— groups, —CO—O—groups, —O—CO—O, —O—CO—NR^(a)—, —NR—CO—O—, —CO—NR^(a)— and/or—NR^(a).CO— groups. Suitable aryl groups include phenyl and naphthylgroups, while suitable heteroaryl groups include pyridine, pyrrole,furan, pyran, imidazole, pyrazole, oxazole, pyridazine, pyrimidine andpurine. Especially suitable linking groups are heteroaryl or,especially, aryl groups, especially phenyl groups. These may be adjacenta further portion of the linking group which is, or contains, a—NR^(a).CO— or —CO.NR^(a)— group, for example an —NH.CO— or —CO.NH—group. Here and elsewhere throughout this Specification, where a groupR^(a) is present, this is preferably a C₁₋₄alkyl, especially a methylgroup or, especially, a hydrogen atom.

Substituents which may be present on an optionally substituted aryl,especially phenyl, or heteroaryl group include for example one or moreof the same or different substituents selected from alkyl (preferablyC₁₋₄alkyl, especially methyl, optionally substituted by OH or CO₂H),CF₃, NR^(a) ₂, —CN, —NO₂, —CO₂R^(a), —COH, —CH₂OH, —COR^(a), —OR^(a),—OCOR^(a), —OCO₂R^(a), —SR^(a), —SOR^(a), —SO₂R^(a), —NR^(a)COR^(a),—NR^(a).CO₂R^(a), —NO, —NR^(a).OH, —CH═N—NR^(a).COR^(a), —N⁺R^(a) ₃,halogen, for example fluorine or chlorine, —C≡CR^(a), and —CH═CR^(a) ₂,in which each R^(a) independently represents a hydrogen atom or an alkyl(preferably C₁₋₆alkyl), aryl (preferably phenyl), or alkyl-aryl(preferably C₁₋₆alkyl-phenyl) group. The presence of electronwithdrawing substituents is especially preferred. Preferred substituentsinclude for example CN, NO₂, —OR^(a), —OCOR^(a), —SR^(a),—NR^(a).COR^(a), —NHOH and —NR^(a).COR^(a), especially CN and NO₂.

Preferably the linker includes one of the above groups adjacent thegrouping F′/F. One preferred group of conjugates and reagents includesthose wherein the conjugate/reagent comprises a linker between thetherapeutic, diagnostic or labelling agent and the grouping of formulaF′/F, which linker includes an optionally substituted aryl or heteroarylgroup immediately adjacent the grouping F′/F; and which linker alsoincludes a —NR^(a).C(O)— or —C(O).NR^(a)— group adjacent said aryl orheteroaryl group; thus having the formula —NR^(a).C(O)-(het)aryl-F′ or—C(O).NR^(a)-(het)aryl-F, wherein R^(a) represents C₁₋₄ alkyl orhydrogen. Especially preferred are conjugates and conjugating reagentswhich include the grouping:

or, especially:

In the above formulae, preferably F has the formula I or II, for exampleIa, Ib, IIa or IIb above, and preferably F′ has the formula III, forexample IIIa, IIIb, or IIIc above.

In an alternative embodiment, conjugates and reagents of the inventioninclude those which comprise a linker between the therapeutic,diagnostic or labelling agent and the grouping of formula F′/F, whichlinker includes an optionally substituted aryl or heteroaryl groupimmediately adjacent the grouping F′/F; and which linker also includes a—NR.C(O)— or —C(O).NR^(b)— group adjacent said aryl or heteroaryl group;thus having the formula —NR.C(O)-(het)aryl-F′ or—C(O).NR^(b)-(het)aryl-F, wherein R^(b) represents a group containing apolyethylene glycol ring.

Subsection (ii). In one embodiment, the linker may contain a degradablegroup, i.e. it may contain a group which breaks under physiologicalconditions, separating the payload from the protein to which it is, orwill be, bonded. Alternatively, it may be a linker that is not cleavableunder physiological conditions. Where a linker breaks underphysiological conditions, it is preferably cleavable under intracellularconditions. Where the target is intracellular, preferably the linker issubstantially insensitive to extracellular conditions (i.e. so thatdelivery to the intracellular target of a sufficient dose of thetherapeutic agent is not prohibited).

Where the linker contains a degradable group, this is generallysensitive to hydrolytic conditions, for example it may be a group whichdegrades at certain pH values (e.g. acidic conditions).Hydrolytic/acidic conditions may for example be found in endosomes orlysosomes. Examples of groups susceptible to hydrolysis under acidicconditions include hydrazones, semicarbazones, thiosemicarbazones,cis-aconitic amides, orthoesters and ketals. Examples of groupssusceptible to hydrolytic conditions include:

In a preferred embodiment, the linker includes

For example, it may include:

The linker may also be susceptible to degradation under reducingconditions. For example, it may contain a disulfide group that iscleavable on exposure to biological reducing agents, such as thiols.Examples of disulfide groups include:

in which R, R′, R″ and R″′ are each independently hydrogen or C₁₋₄alkyl.In a preferred embodiment the linker includes

For example, it may include

The linker may also contain a group which is susceptible to enzymaticdegradation, for example it may be susceptible to cleavage by a protease(e.g. a lysosomal or endosomal protease) or peptidase. For example, itmay contain a peptidyl group comprising at least one, for example atleast two, or at least three amino acid residues (e.g. Phe-Leu,Gly-Phe-Leu-Gly, Val-Ala, Val-Cit, Phe-Lys, Glu-Glu-Glu). For example,it may include an amino acid chain having from 1 to 5, for example 2 to4, amino acids. Another example of a group susceptible to enzymaticdegradation is:

wherein AA represents a protease-specific amino acid sequence, such asVal-Cit.

In a preferred embodiment, the linker includes:

For example, it may include

The linker may carry a single payload D, or more than one group D.Multiple groups D may be incorporated by the use of a branching linker,which may for example incorporate an aspartate or glutamate or similarresidue. This introduces a branching element of formula:

where b is 1, 2 or 3, b=1 being aspartate and b=2 being glutamate, andb=3 representing one preferred embodiment. Each of the acyl moieties inthe above formula may be coupled to a group D. The branching group abovemay incorporate a —CO.CH₂— group, thus:

If desired, the aspartate or glutamate or similar residue may be coupledto further aspartate and/or glutamate and/or similar residues, forexample:

and so on.

In a similar way, the amino acids lysine, serine, threonine, cysteine,arginine or tyrosine or similar residues may be introduced to form abranching group, thus:

in which b is 4 for lysine, and

in which b is 1 for serine.

Similar branching groups may be used to incorporate the cyclic PEG groupinto the linker. So, for example, one of the branching elementsmentioned above, for example an aspartate, glutamate, lysine or serineor similar residue, may be present with one branch leading to a drug Dwhile the other leads to a branch containing the cyclic PEG group. Thevarious linker portions mentioned above may be present at any locationeither before or after a branching group.

As will be apparent, many alternative configurations for the linkerbetween the grouping F/F′ and the payload are possible. One preferredconfiguration may be represented schematically as follows:

in which E represents one of the groups mentioned in subsection (i)above, and B represents one of the groups mentioned in this subsection(ii).

A specific, particularly preferred construction is shown below:

in which F′ and F have the meanings and preferred meanings given above.Particularly preferred examples of such structures are as follows:

Subsection (iii). The linker which connects the therapeutic, diagnosticor labelling agent to the protein or peptide in the conjugates of theinvention or to the functional grouping in the conjugating reagents ofthe invention may contain additional PEG in addition to the cyclic PEGgroup. It may for example contain PEG in the backbone of the linker,shown schematically thus:

In these formulae, p and q represent the number of ethylene glycol unitspresent in the various PEG chains present in the linker of the conjugateor the reagent in addition to the n ethylene glycol units present in thering. For clarity, the PEG units are shown as straight-chain units, butit will be understood that any of the units may include branched chains.

Similarly, here and elsewhere, for clarity, a

group is shown as having only single points of attachment from the ringto the rest of the linker, but it should be understood that except wherethe context requires otherwise, multiple points of attachment asdescribed above are equally possible.

Subsection (iv). The linker which connects the therapeutic, diagnosticor labelling agent to the protein or peptide in the conjugates of theinvention or to the functional grouping in the conjugating reagents ofthe invention may contain two or more cyclic PEG groups. This may beillustrated schematically for two cyclic PEG groups thus:

and obviously more than two cyclic PEG groups may similarly be present.The linker may or may not contain additional PEG in addition to thecyclic PEG groups, as described in subsection (iii) above.

Multiple cyclic PEG groups may be incorporated into the linker using anysuitable method. A cyclic PEG group may for example be introduced byreaction with any reactive grouping present in any of the linkerportions discussed above. Branching groups of the formulae describedabove may be used. For example, in one specific embodiment, two cyclicPEG groups may be incorporated by use of a structure:

Alternatively, branching may be introduced by use of a polyolfunctionality, for example:

˜CH_(s)[(CH₂)_(t)O]_(3-s)˜

in which s is 0, 1 or 2, and t is 1 to 4. For example, in one specificembodiment, three cyclic PEG groups may be incorporated by use of astructure:

In some preferred embodiments of the conjugate, the conjugate includes aportion:

in which W′ represents an electron withdrawing group or a group obtainedby reduction of an electron withdrawing group, and Pr represents aprotein or peptide bonded via nucleophiles Nu,

and which comprises an optionally substituted aryl or heteroaryl groupimmediately adjacent the group of formula IIIa; and which linker alsoincludes a —NR^(a).C(O)— or —C(O).NR^(a)— group adjacent said aryl orheteroaryl group; wherein R^(a) represents C₁₋₄ alkyl or hydrogen;

the protein or peptide is an antibody (e.g. an anti-CD30 antibody suchas brentuximab);

the linker comprises a group

and

the therapeutic, diagnostic or labelling agent is a cytotoxic agent(e.g. an auristatin, maytansine or duocarmycin).

In some preferred embodiments of the conjugate, the conjugate includes aportion:

in which W′ represents an electron withdrawing group or a group obtainedby reduction of an electron withdrawing group, and Pr represents aprotein or peptide bonded via nucleophiles Nu,

and which comprises an optionally substituted aryl or heteroaryl groupimmediately adjacent the group of formula IIIa; and which linker alsoincludes a —NR^(a).C(O)— or —C(O).NR^(a)— group adjacent said aryl orheteroaryl group; wherein R^(a) represents C₁₋₄ alkyl or hydrogen;

the protein or peptide is an antibody (e.g. an anti-CD30 antibody suchas brentuximab); the linker comprises a group

wherein the ring contains from 2 to 20 —CH₂CH₂—O— units (e.g. 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20);

the linker comprises a degradable group which is

the linker comprises a branching group which is an aspartate, glutamate,lysine or serine residue to incorporate the cyclic PEG group into thelinker; and

the therapeutic, diagnostic or labelling agent is monomethyl auristatinE (MMAE).

In some preferred embodiments of the conjugate, the conjugate includes aportion:

in which W′ represents an electron withdrawing group or a group obtainedby reduction of an electron withdrawing group, and Pr represents aprotein or peptide bonded via nucleophiles Nu,

and which comprises an optionally substituted aryl or heteroaryl groupimmediately adjacent the group of formula IIIa; and which linker alsoincludes a —NR^(a).C(O)— or —C(O).NR^(a)— group adjacent said aryl orheteroaryl group; wherein R^(a) represents C₁₋₄ alkyl or hydrogen;

the protein or peptide is an antibody (e.g. an anti-CD30 antibody suchas brentuximab); the linker comprises a group or;

the linker comprises a degradable group which is

the linker comprises a branching group which is a glutamate residue toincorporate the cyclic PEG group into the linker; and

the therapeutic, diagnostic or labelling agent is monomethyl auristatinE (MMAE).

In some preferred embodiments, the conjugate has the formula:

wherein D is a cytotoxic agent (e.g. an auristatin, maytansine orduocarmycin);

represents a ring which includes at least two ethylene glycol,˜(CH₂—CH₂—O—)˜, units; F′ is a group of formula

and

Pr represents a protein or peptide bonded via nucleophiles Nu, which isan antibody (e.g. an anti-CD30 antibody such as brentuximab).

In some preferred embodiments, the conjugate has the formula:

wherein D is monomethyl auristatin E;

represents a group or

wherein the ring contains from 2 to 20 —CH₂CH₂—O— units (e.g. 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20);

F′ is a group of formula

and

Pr represents a protein or peptide bonded via nucleophiles Nu, which isan antibody (e.g. an anti-CD30 antibody such as brentuximab).

In some preferred embodiments, the conjugate has the formula:

wherein D is monomethyl auristatin E;

represents

F′ is a group of formula

and

Pr represents a protein or peptide bonded via nucleophiles Nu, which isan antibody (e.g. an anti-CD30 antibody such as brentuximab).

Pharmaceutical Compositions and Utility

Conjugates according to the invention in which the payload is atherapeutic agent find utility in the treatment of various medicalconditions depending on the nature of the payload. Typically the payloadwill be a cytotoxic agent and the invention finds utility in thetreatment of cancer. Accordingly, the invention further provides aconjugate according to the present invention, particularly one in whichthe payload is a therapeutic agent and specifically a conjugate which isan antibody-drug conjugate, together with a pharmaceutically acceptablecarrier, and optionally together with a further active ingredient. Theinvention further provides the use of such a conjugate in therapy, andfinds utility in a method of treatment of a patient which comprisesadministering a conjugate or a pharmaceutical composition according tothe invention to the patient. The invention further provides the use ofa conjugate according to the invention in the manufacture of amedicament for the treatment of, for example, cancer.

BRIEF DESCRIPTION OF THF DRAWINGS

FIG. 1 shows the results of Example 8.

FIGS. 2A-2B show the results of mouse xenograft studies, with FIG. 2Ashowing (a) a plot of mean tumour volume±standard error over time inCB17-SCID mice following administration of vehicle or Adcetris®(brentuximab vedotin) (comparator) at 1 mg/kg on day 12 following tumourimplantation; and FIG. 2B showing (b) plots of individual tumour volumesover time in CB17-SCID mice following administration of vehicle orAdcetris® (brentuximab vedotin) (comparator) at 1 mg/kg on day 12following tumour implantation. FIGS. 3A-3B show the results of mousexenograft studies, with FIG. 3A showing (a) a plot of mean tumourvolume±standard error over time in CB17-SCID mice followingadministration of vehicle or conjugate 32 (comparator) at 0.8 mg/kg onday 12 following tumour implantation; and FIG. 3B showing (b) plots ofindividual tumour volumes over time in CB17-SCID mice followingadministration of vehicle or conjugate 32 (comparator) at 0.8 mg/kg onday 12 following tumour implantation.

FIGS. 4A-4B show the results of mouse xenograft studies, with FIG. 4Ashowing (a) a plot of mean tumour volume±standard error over time inCB17-SCID mice following administration of vehicle or conjugate 14 at0.8 mg/kg on day 12 following tumour implantation; and FIG. 4B showing(b) plots of individual tumour volumes over time in CB17-SCID micefollowing administration of vehicle or conjugate 14 at 0.8 mg/kg on day12 following tumour implantation.

FIGS. 5A-5B show the results of mouse xenograft studies, with FIG. 5Ashowing (a) a plot of mean tumour volume±standard error over time inCB17-SCID mice following administration of vehicle or conjugate 31 at0.8 mg/kg on day 12 following tumour implantation; and FIG. 5B showing(b) plots of individual tumour volumes over time in CB17-SCID micefollowing administration of vehicle or conjugate 31 at 0.8 mg/kg on day12 following tumour implantation.

The following Examples illustrate the invention.

Example 1: Synthesis of Conjugation Reagent 1 Comprising the AuristatinCytotoxic Payload, MAE

Step 1: Synthesis of Compound 2

To a stirred solution of diethanolamine (2.5 g) and triethylamine (6.05g) in dichloromethane (15 mL) was slowly added a solution of tosylchloride (3.8 g) in dichloromethane (15 mL) at room temperature. After 2h, water (25 mL) was added to the reaction mixture and the product wasextracted with dichloromethane (5×30 mL). The combined organic extractswere dried over magnesium sulfate, the solution was then filtered andthe volatiles removed in vacuo to yield compound 2 as a white solid (4.7g). ¹H NMR (400 MHz, CDCl₃) δ_(H) 7.68 (d, J=8.3 Hz, 2H), 7.30 (d, J=8.3Hz, 2H), 3.84 (t, J=5.0 Hz, 4H), 3.56 (s, 2H), 3.24 (t, J=5.0 Hz, 4H),2.41 (s, 3H). m/z [M+Na]⁺ (282, 95%), [M+H]⁺ (260, 100%).

Step 2: Synthesis of Compound 3

A solution of compound 2 (176 mg) in anhydrous THF (2 mL) was addeddropwise over a period of 1 h to a solution of sodium hydride (80 mg,60% dispersion in mineral oil) in anhydrous THF (8 mL) at roomtemperature. After stirring for 1 h, a solution of hexaethyleneglycoldi-p-toluenesulfonate (400 mg) in anhydrous THF (2 mL) was added over aperiod of 2 h and the reaction mixture was stirred at room temperaturefor 72 h. Water (30 mL) was added and the THF was removed in vacuo. Theaqueous solution was extracted with chloroform (4×25 mL), the organicphases were combined and dried over magnesium sulfate before thesolution was filtered and concentrated in vacuo. The residue was thenpurified by reverse phase C18-column chromatography eluting with bufferA (v/v): water:55% acetonitrile:0.05% trifluoroacetic acid and buffer B(v/v):acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v).The organic solvent was removed in vacuo and the aqueous solvent wasremoved by lyophilisation to give compound 3 as a colourless oil (78mg). ¹H NMR (400 MHz, CDCl₃) δ_(H) 7.68 (d, J=8.3 Hz, 2H), 7.26 (d,J=8.3 Hz, 4H), 3.67-3.58 (m, 24H), 3.58-3.53 (m, 4H), 3.38 (t, J=6.0 Hz,4H), 2.40 (s, 3H). m/z [M+Na]*(528, 80%), [M+H]⁺ (506, 50%).

Step 3: Synthesis of Compound 4

To a solution of compound 3 (78 mg) in anhydrous THF (6 mL) was addedlithium aluminium hydride (1.13 mL, 1 M solution in THF) and thesolution was heated at reflux for 16 h before the reaction mixture wascooled to 0° C. and quenched by the dropwise addition of water. Thesuspension was filtered and the precipitate washed withchloroform:ethanol (9:1 v/v, 5×6 mL). The filtrate and washings werecombined and concentrated in vacuo to give compound 4 as a colourlessoil (50 mg). m/z [M+Na]⁺ (374, 70%), [M+H]⁺ (352, 100%).

Step 4: Synthesis of Compound 5

To a solution of Fmoc-Glu-(OH)—OAll (48 mg) in DMF (1.0 mL) was addedHATU (110 mg) and the solution was stirred for 30 min at 0° C. To thiswas added a solution of Val-Cit-PAB-MMAE.TFA salt (Levena Biopharma, 120mg) and NMM (32 μL) in DMF (1 mL). The reaction mixture was stirred at0° C. for 2.5 h. The solvent was concentrated in vacuo and the crude wasdissolved in DMF (1.5 mL) before NN (32 μL) was added.Tetrakis(triphenylphosphine)palladium(0) (45 mg) was added to thereaction mixture which was then stirred at room temperature for 20 h.The reaction solution was concentrated in vacuo and the residue purifiedby reverse phase C18-column chromatography eluting with buffer A (v/v):water:5% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v):acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). Theorganic solvent was removed in vacuo and the aqueous solvent was removedby lyophilisation to give compound 5 as a white solid (98.0 mg). m/z[M+H]⁺ (1475 Da, 100%), [M+2H]²⁺ (738, 50%).

Step 5: Synthesis of Compound 6

To a solution of compound 5 (15 mg) in DMF (0.5 mL) at 0° C. was addedHATU (4.3 mg). The solution was stirred for 20 min at 0° C. before NMM(1.3 μL) was added and the solution was stirred for a further 10 min. Toa separate solution of compound 4 (5 mg) in DMF (0.3 ml) was added NMM(1.3 μL) and the solution was stirred at room temperature for 30 min.The two solutions were then combined and additional quantities of HATU(4.3 mg) and NMM (1.3 μL) were added to the combined solution which wasstirred for 16 h at room temperature. The reaction solution wasconcentrated in vacuo to give crudeFmoc-L-Glu-[Val-Cit-PAB-MMAE]-aza-24-crown-8 (18 mg). m/z [M+Na]⁺ (1831,20%), [M+H]⁺ (1809, 20%), [M+2H]²⁺ (905, 100%). To a solution ofFmoc-L-Glu-[Val-Cit-PAB-MMAE]-aza-24-crown-8 (18 mg) in DMF (0.4 mL) wasadded piperidine (5 μL) and the solution was stirred for 20 min at roomtemperature. Additional piperidine (5 μL) was added and the solution wasstirred for a further 25 min at room temperature. The reaction solutionwas concentrated in vacuo and the residue purified by reverse phaseC18-column chromatography eluting with buffer A (v/v): water:55%acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v):acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). Theorganic solvent was removed in vacuo and the aqueous solvent was removedby lyophilisation to give compound 6 as a colourless oil (8.5 mg). m/z[M+Na]⁺ (1609, 10%), [M+H]⁺ (1587, 20%), [M+2H]²⁺ (794, 100%)

Step 6: Synthesis of Compound 2

To a stirred solution of4-[2,2-bis[(p-tolylsulfonyl)-methyl]acetyl]benzoic acid (1.5 g, NatureProtocols, 2006, 1(54), 2241-2252) in DMF (70 mL) was addedalpha-methoxy-omega-mercapto hepta(ethylene glycol) (3.2 g) andtriethylamine (2.5 mL). The resulting reaction mixture was stirred underan inert nitrogen atmosphere at room temperature. After 19 h, volatileswere removed in vacuo. The resulting residue was dissolved in water (2.4mL) and purified by reverse phase C18-column chromatography eluting withbuffer A (v/v): water:55% acetonitrile:0.05% trifluoroacetic acid andbuffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to0:100 v/v). The organic solvent was removed in vacuo and the aqueoussolvent was removed by lyophilisation to give compound 7 as a thickclear colourless oil (1.77 g). m/z [M+H]⁺ 901.

Step 7: Synthesis of Reagent 8

To a stirred solution of 7 (1.32 g) in methanol:water (18 mL, 9:1 v/v)at room temperature was added Oxone® (2.7 g). After 2.5 h, the volatileswere removed in vacuo and water was azeotropically removed withacetonitrile (2×15 mL). The resulting residue was dissolved indichloromethane (3×10 mL), filtered through a column of magnesiumsulfate and washed with dichloromethane (2×7 mL). The eluent andwashings were combined and the volatiles were removed in vacuo to give athick clear pale yellow oil (1.3 g). A portion of the residue (700 mg)was dissolved in water:acetonitrile (1.5 mL, 3:1 v/v), and purified byreverse phase C18-column chromatography eluting with buffer A (v/v):water:5% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v):acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). Theorganic solvent was removed in vacuo and the aqueous solvent was removedby lyophilisation to give reagent 8 as a thick clear colourless oil (524mg). m/z [M+H]⁺ 965.

Step 8: Synthesis of Reagent 1

To a solution of reagent 8 (6 mg) in DMF (0.3 mL) at 0° C. was addedHATU (2.3 mg) The solution was stirred at 0° C. for 20 min before NMM(0.6 μL) was added and the solution was stirred for a further 10 min. Toa separate solution of compound 6 (8.5 mg) in DMF (0.3 mL) was added NMM(0.6 μL) and the solution was stirred at room temperature for 30 min.The two solutions were then combined and additional quantities of HATU(2.3 mg) and NMM (0.6 μL) were added and the solution was stirred atroom temperature for 1 h. Further quantities of HATU (1.2 mg) and NMM(0.3 μL) were then added and the solution stirred at room temperaturefor 1.5 h. The reaction solution was concentrated in vacuo and theresidue purified by reverse phase C18-column chromatography eluting withbuffer A (v/v): water:55% acetonitrile:0.05% trifluoroacetic acid andbuffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to0:100 v/v). The organic solvent was removed in vacuo and the aqueoussolvent was removed by lyophilisation to give reagent 1 as a colourlessoil (10.7 mg). m/z [M+Na]⁺ (2557, 5%), [M+2H]²⁺ (1268, 40%), [M+3H]³⁺(845, 45%).

Example 2: Synthesis of Conjugation Reagent 9 Comprising the AuristatinCytotoxic Payload, MMAE

Step 1: Synthesis of Compound 10

To a solution of tosyl chloride (307 mg) in dichloromethane (5 mL) wasadded a solution of dodecaethylene glycol (400 mg), triethylamine (255μL) and DMAP (13 mg) in dichloromethane (5 mL) and the combined solutionwas stirred at room temperature for 16 h. Additional DMAP (13 mg) andtriethylamine (255 μL) was added and the reaction mixture was allowed tostir at room temperature for a further 24 h. The reaction solution wasconcentrated in vacuo and the residue purified by reverse phaseC18-column chromatography eluting with buffer A (v/v): water:5%acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v):acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). Theorganic solvent was removed in vacuo and the aqueous solvent was removedby lyophilisation to give compound 10 as a colourless oil (287 mg). ¹HNMR (400 MHz, CDCl₃) δ_(H) 7.78 (d, J=8.5 Hz, 4H), 7.32 (d, J=8.5 Hz,4H), 4.13 (t, J=4.9 Hz, 4H), 3.66 (t, J=4.9 Hz, 4H), 3.62 (br. s, 24H),3.61-3.58 (m, 8H), 3.56 (s, 8H), 2.42 (s, 6H). m/z [M+Na]⁺ (877, 100%),[M+H]⁺ (855, 75%).

Step 2: Synthesis of Compound 11

A solution of compound 2 (85 mg) in anhydrous THF (2 mL) was addeddropwise over a period of 1 h to a solution of sodium hydride (40 mg,60% dispersion in mineral oil) in anhydrous THF (8 mL) at roomtemperature. After stirring for 1 h, a solution of compound 10 (280 mg)in anhydrous THF (20 mL) was added over a period of 2 h and the reactionmixture was stirred at room temperature for 96 h. Water (30 mL) wasadded and the THF was removed in vacuo. The aqueous solution wasextracted with chloroform (4×25 mL) and then chloroform:isopropanol (9:1v/v, 2×25 mL). The organic phases were combined and dried over magnesiumsulfate before the solution was filtered and concentrated in vacuo. Theresidue was then purified by reverse phase C18-column chromatographyeluting with buffer A (v/v): water:55% acetonitrile:0.05%trifluoroacetic acid and buffer B (v/v):acetonitrile:0.05%trifluoroacetic acid (100:0 v/v to 0:100 v/v). The organic solvent wasremoved in vacuo and the aqueous solvent was removed by lyophilisationto give compound 11 as a colourless oil (58 mg). ¹H NMR (400 MHz, CDCl₃)δ_(H) 7.68 (d, J=8.4 Hz, 2H), 7.27 (d, J=8.4 Hz, 4H), 3.66-3.52 (m,52H), 3.35 (t, J=6.3 Hz, 4H), 2.40 (s, 3H). m/z [M+Na]⁺ (792, 100%),[M+H]⁺ (770, 55%).

Step 3: Synthesis of Compound 12

To a solution of compound 11 (54 mg) in anhydrous THF (3 mL) was addedlithium aluminium hydride (513 μL, 1 M solution in THF) and the solutionwas heated at reflux for 4 h before the reaction mixture was cooled to0° C. and quenched by the dropwise addition of water. The suspension wasfiltered and the precipitate washed with chloroform:ethanol (9:1 v/v,3×6 mL). The filtrate and washings were combined and concentrated invacuo to give compound 12 as a white solid (34 mg). m/z [M+Na]⁺ (639,10%), [M+H]⁺ (617, 100%).

Step 4: Synthesis of Compound 13

To a solution of compound 5 (25 mg) in DMF (0.4 mL) at 0° C. was addedHATU (7 mg). The solution was stirred for 20 min at 0° C. before NN (2μL) was added and the solution was stirred for a further 10 min. To aseparate solution of compound 12 (17 mg) in DMF (0.4 ml) was added NMM(2 μL) and the solution was stirred at room temperature for 30 min. Thetwo solutions were then combined and additional quantities of HATU (7mg) and NMM (2 μL) were added to the combined solution which was stirredfor 1 h at room temperature. Further HATU (7 mg) and NMM (2 μL) wereadded and the solution stirred for 0.5 h before the reaction solutionwas concentrated in vacuo to give crudeFmoc-L-Glu-[Val-Cit-PAB-MMAE]-aza-42-crown-14 (35 mg). m/z [M+Na]⁺(2095, 25%), [M+H]⁺ (2073, 15%, [M+2H]²⁺ (1037, 100%). To a solution ofcrude Fmoc-L-Glu-[Val-Cit-PAB-MMAE]-aza-42-crown-14 (35 mg) in DMF (0.5mL) was added piperidine (10 μL) and the solution was stirred for 30 minat room temperature. Additional piperidine (10 μL) was added and thesolution was stirred for a further 30 min at room temperature. Thereaction solution was concentrated in vacuo and the residue purified byreverse phase C18-column chromatography eluting with buffer A (v/v):water:55% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v):acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). Theorganic solvent was removed in vacuo and the aqueous solvent was removedby lyophilisation to give compound 13 as a colourless oil (22 mg). m/z[M+Na]⁺ (1873, 10%), [M+H]⁺ (1851, 50%), [M+2H]²⁺ (926, 100%).

Step 5: Synthesis of Reagent 9

To a solution of reagent 8 (11.5 mg) in DMF (0.3 mL) at 0° C. was addedHATU (3.3 mg) The solution was stirred at 0° C. for 20 min before NN(0.9 μL) was added and the solution was stirred for a further 10 min. Toa separate solution of compound 13 (15 mg) in DMF (0.3 mL) was added NMM(0.9 μL) and the solution was stirred at room temperature for 30 min.The two solutions were then combined and additional quantities of HATU(3.3 mg) and NMM (0.9 μL) were added and the solution was stirred atroom temperature for 1 h. Further quantities of HATU (3.3 mg) and NMM(0.9 μL) were added and the solution stirred at room temperature for 1h. HATU (3.3 mg) and NMM (0.92 μL) were again added and the solution wasstirred at room temperature for 2 h before the reaction solution wasconcentrated in vacuo and the residue purified by reverse phaseC18-column chromatography eluting with buffer A (v/v): water:5%acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v):acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). Theorganic solvent was removed in vacuo and the aqueous solvent was removedby lyophilisation to give reagent 9 as a colourless oil (11 mg). m/z[M+Na]⁺ (2822, 10%), [M+H]⁺ (2800, 20%), [M+2H]²⁺ (1400, 80%), [M+3H]³⁺(933, 100%).

Example 3: Conjugation of Reagents 1 and 17 (Comparative) to Brentuximaband Anti-PSMA Antibodies to Produce Antibody Drug Conjugates (ADCs) 4and 18 (Comparative) and 21 and 22 (Comparative), Respectively, with DAR4

Conjugation reagents 1 and 17 were conjugated to Brentuximab oranti-PSMA antibody, giving rise to ADCs 14, 18, 21 and 22 respectivelyusing methods analogous to those described in WO2014064423 andWO2014064424. Briefly, antibody (Brentuximab or anti-PSMA antibody) at aconcentration of 5.2 mg/mL in 20 mM sodium phosphate, pH 7.5 (containing150 mM NaCl and 20 mM EDTA) was heated to 40° C. in a heating block for15 min. TCEP (6 eq. per mAb) was added to the mAb solution, mixed gentlyand incubated at 40° C. for 1 h before being allowed to cool to 22° C.Conjugation reagents were dissolved in MeCN or DMF to give a 3 mMsolution. The reduced mAb solution was diluted to 4.2 mg/mL with 20 mMsodium phosphate, pH 7.5 (containing 150 mM NaCl and 20 mM EDTA).Conjugation reagent (5.6 eq. per mAb) was added to the mAb solution, thereaction was mixed gently and incubated at 22° C. for 22 h. After thisthe reaction was treated with 50 mM N-acetyl-L-cysteine (20 eq. overreagent) at 22° C. for 1 h. The crude conjugation mixture was analysedby hydrophobic interaction chromatography (HIC). The crude reaction wasmixed with an equal volume of 50 mM sodium phosphate, pH 7 (4 M NaCl)and the resulting solution was loaded onto a ToyoPearl Phenyl-650S HICcolumn equilibrated with 50 mM sodium phosphate, pH 7 (2 M NaCl). TheADC was eluted from the column with a gradient of 50 mM sodiumphosphate, pH 7 (20% isopropanol). Fractions containing DAR 4 ADC werepooled and concentrated (Vivaspin 20, 10 kDa PES membrane). Theconcentrated sample was buffer exchanged into PBS, pH 7.1-7.5, andsterile filtered (0.22 μm PVDF membranes). DAR assignments were based onA248/A280 absorption ratios. The average DAR of conjugates wascalculated from the relative peak areas of individual DAR speciesfollowing HIC analysis at 280 nm.

Example 4: Synthesis of Conjugation Reagent 15 (Comparative) Comprisingthe Auristatin Cytotoxic Payload, MMAE

Conjugation reagent 5, which contains a maleimide functional grouping,was synthesised as described within WO2015057699.

Example 5: Synthesis of Conjugation Reagent 17 (Comparative) Comprisingthe Auristatin Cytotoxic Payload, MMAE

Step 1: Synthesis of Compound 19

A solution of Fmoc-L-Glu-(OtBu)-OH (36 mg) in DMF (2 mL) was cooled to0° C. under an argon atmosphere and(benzotriazol-1-yloxy)tris-(dimethylamino) phosphoniumhexafluorophosphate (BOP) (41 mg) was added, followed byNH₂—PEG(24u)-OMe (100 mg) and DIPEA (19 μL). The solution was allowed towarm to room temperature and after 22 h the volatiles were removed invacuo. The resulting residue was dissolved in dichloromethane (1 mL) andpurified by normal phase column chromatography eluting withdichloromethane:methanol (100:0 v/v to 80:20 v/v). The organic solventwas removed in vacuo to give Fmoc-L-Glu-[OtBu]-[PEG(24u)-OMe] as acolourless oil (84 mg). Piperidine (49 μL) was added to a solution ofcompound Fmoc-L-Glu-[OtBu]-[PEG(24u)-OMe] (74 mg) in DMF (2 mL) under anargon atmosphere and the resulting solution stirred at room temperaturefor 22 h, after which the volatiles were removed in vacuo. The resultingresidue was triturated with hexane (3×0.7 mL). The organic solvent wasdecanted each time and the resulting residue dried in vacuo to givecompound 19 as a white solid (61 mg). m/z [M+H]⁺ (1274, 70%), [M+2H]²⁺(638, 100%).

Step 2: Synthesis of Reagent 20

To a solution of reagent 8 (26.6 mg) in DMF (550 μL) cooled to 0° C.under an argon atmosphere was added HATU (10.5 mg) and the solutionstirred for 0.5 h at 0° C. To this was added a solution of compound 19(32 mg) in DMF (550 μL). The resulting solution was stirred for 5 min at0° C. before the addition of NMM (2.9 μL) and HATU (10.5 mg). Thereaction solution was allowed to stir at 0° C. for 2 h before beingwarmed to room temperature and stirred for a further 3.5 h. After thistime the volatiles were removed in vacuo. The resulting residue wasdissolved in water:acetonitrile (1.2 mL, 1:1 v/v), and purified byreverse phase C18-column chromatography eluting with buffer A (v/v):water:55% acetonitrile:0.1% formic acid and buffer B (v/v):acetonitrile:0.1% formic acid (100:0 v/v to 0:100 v/v). The organicsolvent was removed in vacuo and the aqueous solvent removed bylyophilisation to givebis-mPEG(7u)sulfone-propanoyl-benzamide-L-Glu-[OtBu]-[PEG(24u)-OMe] as acolourless oil (30.5 mg). ¹H NMR (400 MHz, MeOH—O₄) 8.19 (2H, d), 8.04(2H, d), 4.83-4.71 (1H, m), 4.58 (1H, dd), 3.92-3.83 (6H, m), 3.78-3.56(140H, m), 3.57-3.51 (6H, m), 3.40 (4H, dd), 3.36 (3H, s), 3.35 (6H, s),2.41 (2H, t), 2.24-2.13 (1H, m), 2.10-1.98 (1H, m), 1.45 (9H, s). m/z[M+Na]⁺ (2243, 50%), [M+H]⁺ (2221, 40%), [M+Na+2H]³⁺ (747, 100%). Asolution ofbis-mPEG(7u)sulfone-propanoyl-benzamide-L-Glu-[OtBu]-[PEG(24u)-OMe] (30mg) in dichloromethane (2 mL) under an argon atmosphere was cooled to 0°C. to which trifluoroacetic acid (500 μL) was added and the resultingsolution stirred for 1.5 h. The reaction mixture was allowed to warm toroom temperature and stirred for a further 1 h. After this time thevolatiles were removed in vacuo. The resulting residue was dissolved inwater:acetonitrile (0.6 mL, 1:1 v/v), and purified by reverse phaseC18-column chromatography eluting with buffer A (v/v): water:55%acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v):acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). Theorganic solvent was removed in vacuo and the aqueous solvent removed bylyophilisation to give reagent 20 as a colourless oil (20 mg). ¹H NMR(400 MHz, MeOH-δ₄) 8.19 (2H, d), 8.04 (2H, d), 4.81-4.72 (1H, m), 4.59(1H, dd), 3.92-3.84 (6H, m), 3.67-3.50 (146H, m), 3.40 (4H, dd), 3.36(3H, s), 3.35 (6H, s), 2.48 (2H, t), 2.26-2.15 (1H, m), 2.15-2.03 (1H,m). m/z [M+2H]²⁺ (1083, 60%), [M+2H+Na]³⁺ (729, 100%).

Step 3: Synthesis of Conjugation Reagent 17

A solution of compound 20 (12.4 mg) in DMF (500 μL) was cooled to 0° C.under an argon atmosphere. HATU (2.4 mg) was added and the solutionstirred for 0.5 h at 0° C. To this was added a solution ofVal-Cit-PAB-MMAE TFA salt (7.8 mg) and NN (0.7 μL) in DMF (500 μL),which had been stirred at room temperature for 0.5 h. After 5 min, anadditional amount of HATU (1.2 mg) and NMM (0.4 μL) was added and thereaction mixture stirred at room temperature. After 2 h, an additionalamount of HATU (1.2 mg) and NMM (0.4 μL) was added and the reactionmixture stirred at room temperature. After a further 1 h, the reactionsolution was concentrated in vacuo and purified by reverse phaseC18-column chromatography eluting with buffer A (v/v): water:55%acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v):acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). Theorganic solvent was removed in vacuo and the aqueous solvent removed bylyophilisation to give reagent 17 as a colourless oil. m/z [M+H]⁺ (3270,12%), [M+2H]²⁺ (1636, 50%), [M+3H]³⁺ (1091, 100%).

Example 6: Conjugation of Reagent 15 (Comparative) to Brentuximab toProduce Antibody Drug Conjugate 16 (Comparative) with DAR 8

Conjugation reagent 1 was conjugated to Brentuximab, giving rise to ADC16 using the methods described within WO2015057699, U.S. Pat. No.7,090,843, Lyon et al., (2015) Nature Biotechnology, 33(7) p733-736 andLyon et al., (2012), Methods in Enzymology, Volume 502, p123-137.Briefly, Brentuximab in 20 mM sodium phosphate buffer, pH 6.5 (150 mMNaCl, 20 mM EDTA) was reduced with TCEP (6 eq.) at 40° C. for 1 h.Conjugation of the reduced antibody with 2.0 molar equivalents ofreagent 15 per free thiol was then performed. Reagent 15 was added tothe mAb to give a final antibody concentration of 4 mg/mL. The solutionwas mixed gently and incubated at 22° C. for 2 h. After 2 h additionalreagent 15 (0.2 molar equivalents) was added and the mixture wasincubated for a further 1 h at 22° C. Excess reagent 15 was quenchedwith N-acetyl-L-cysteine (20 eq. over reagent) and the crude reactionwas purified using a 1 mL ToyoPearl Phenyl-650S HIC column equilibratedwith 50 mM sodium phosphate, pH 7 (2 M NaCl). The ADC was eluted fromthe column with a gradient of 50 mM sodium phosphate, pH 7 (20%isopropanol). Fractions containing ADC were pooled and concentrated(Vivaspin20, 10 kDa PES membrane) to give an average DAR 8 product. Theconcentrated sample was buffer exchanged into DPBS, pH 7.1-7.5 andsterile filtered.

ADC 16 was difficult to purify and characterise due to the heterogeneityof the reaction products (number of DAR variants), leading to poorresolution of the individual DAR species by preparative HIC. Resultsshowed that the final reaction product contained significant quantitiesof DAR species both greater than and less than DAR 8. Purifying the DAR8species completely from these higher and lower than DAR8 species wouldresult in significantly lower yields of DAR8 in the final product.

Example 7: Comparison of Antibody Drug Conjugates 14 and 16(Comparative) by Thermal Stress Test

ADC samples 14 and 16 were each prepared at 0.5 mg/mL by dilution withDPBS pH 7.1-7.5.

The two ADC samples were incubated at 65° C. for 30 min followed byincubation in an ice bath for 5 min before Size Exclusion Chromatography(SEC). SEC was performed using a TOSOH Bioscience TSK gel Super SW 3000column. UV absorbance at 280 nm was monitored during an isocraticelution with a 0.2 M potassium phosphate buffer, pH 6.8 (0.2 M potassiumchloride and 15% isopropanol).

Tables 1a and 1b below show the conformations of ADCs 14 and 16 beforeand after thermal stress test, as measured by the area under the curveof each peak by Abs 280 nm, following SEC.

The results in Tables 1a and 1b show that ADC 14 remains in anon-aggregated state to a much greater extent than ADC 16 followingthermal stress test. In addition, the results also show that 16dissociates into lighter molecular weight components to a greater extentthan conjugate 14.

TABLE 1a ADC conformation before thermal stress test (% of total ADC) 1416 Non-aggregated 98.1 97.4 Aggregated 1.9 1.8 Dissociated 0 0.7

TABLE 1b ADC conformation after thermal stress test (% of total ADC) 1416 Non-aggregated 62.0 11.1 Aggregated 37.4 71 Dissociated 0.6 17.9

Example 8: Comparison of Average DARs for ADCs 14 and 16 (Comparative)Following Incubation within Human Serum

ADCs 14 and 16 were diluted to 0.1 mg/mL in human serum, 88% (v/v) serumcontent. Each solution was immediately sub-aliquoted into 4×0.5 mLlow-bind Eppendorf tubes. Two of the Eppendorf tubes, corresponding tothe ‘0’ time points were immediately transferred to the −80° C. freezer,while the remaining samples were incubated at 37° C. for 6 d. After 6 d,the samples were removed from the freezer and/or the incubator, theconjugates purified by affinity capture (CD30-coated magnetic beads),and analysed using hydrophobic interaction chromatography (HIC). CD30affinity capture and HIC for average DAR determination were carried outas described:

CD30 affinity capture for average DAR determination. Affinity capturewas performed using streptavidin coated magnetic beads(Dynabeads-Streptavidin T1, Life Technologies). CD30 (Recombinant humanCD30, Sino Biological Inc.) was biotinylated and immobilized on beadsthrough streptavidin-biotin binding and finally blocked using skimmedmilk peptides. 500 μL of the plasma sample in PBS was added to theCD30-coated beads and incubated overnight at 4° C. and finally washedusing PBS. Captured antibodies were eluted using acidic elution bufferfor 5 min at 4° C. The eluate was subsequently neutralized to pH 7 usingsodium acetate buffer, pH 8. Eluted samples were further mixed with HICloading buffer and analysed using hydrophobic interaction chromatographywith UV detection (HIC-UV).

Hydrophobic interaction chromatography for average DAR determination.Affinity captured ADCs were analysed using hydrophobic interactionchromatography in order to determine the average drug to antibody ratio(DAR). The method consisted of a linear gradient from 100% buffer A (50mM sodium phosphate pH 7.0, 1.5 M ammonium sulfate) to 100% buffer B (50mM sodium phosphate pH 7.0, 20% isopropanol) in 30 min using a TOSOH TSKgel Butyl-NPR HIC separation column with detection at 280 nm.

FIG. 1 shows that after 6 days at 37° C. in human serum, conjugate 16has lost much of its cytotoxic payload, whereas conjugate 14 remainslargely unchanged, as indicated by the reduction in average DAR value ofthe sample. This indicates that conjugates of the invention haveimproved stability.

Example 9: Analysis of DAR4 ADCs 14 and 18 (Comparative) and 21 and 22(Comparative), by In Vitro Cell Viability Assay

Loss of tumour cell viability following treatment with cytotoxic drugsin vitro can be measured by growing cell lines in the presence ofincreasing concentrations of drug and quantifying the loss ofproliferation or metabolic activity using Cell-Titer Glo® Luminescentreagent (Promega). The protocol for PSMA and CD30 overexpressing celllines describes cell seeding, drug treatment and determination of thecell viability in reference to untreated cells based on ATP synthesis,which is directly correlated to the number of cells present in the well.

The cell lines listed in Table 2 were maintained following thesuppliers' recommendations.

TABLE 2 Cell line Antigen Source Growth medium LNCaP clone FGC PSMAECACC, Cat. RPMI-1640 medium 89110211 (Life Technologies ®), C4-2 PSMA10% fetal bovine serum, Karpas-299 CD30 Dr Abraham 100 U/mL Penicillinand Karpas at the 100 ug/mL Streptomycin University of Cambridge

Adherent PSMA-positive LNCaP and C4-2 cells were detached with TrypLEand resuspended in complete medium. Cells were counted using disposableNeubauer counting chambers and cell density adjusted to 10×10⁴ cells/mLfor LNCaP and 2×10⁴ cells/mL for C4-2, respectively. The cells wereseeded (100 μL/well) into poly-D-Lysine coated opaque-walled 96-wellwhite plates and incubated for 24 h at 37° C. and 5% CO₂.

Human T cell lymphoma Karpas 299 cells were counted and adjusted to acell density of 5×10⁴ cells/mL in complete growth medium. Cells wereseeded (50 μL/well) into opaque-walled 96-well plates and incubated for24 h at 37° C. and 5% CO₂.

Eight point serial dilutions of each compound were prepared in therelevant culture medium. Cells were treated with the compounds andconcentration ranges are specified in Table 3.

TABLE 3 Cell line Drug-conjugate Concentration range Karpas 299 14   1nM-0.06 pM Karpas 299 18 (comparative) 0.5 nM-0.2 pM LNCaP & C4-2 21  10nM-5 pM LNCaP & C4-2 22 (comparative)  10 nM-5 pM LNCaP & C4-2 MMAE(free drug)  10 nM-5 pM

The medium from the plate containing the adherent LNCaP or C4-2 cellswas removed and replaced by 100 μL/well of the 1× serially dilutedcompounds. Karpas-299 cells were treated by addition of 50 μL/well ofthe 2× serially diluted compounds. The cells were then incubated at 37°C. and 5% CO₂ for a further 96 h.

The cell viability assay was carried out using the Cell-Titer Glo®Luminescence reagent, as described by the manufacturer's instructions,(Promega Corp. Technical Bulletin TB288; Lewis Phillips G. D, Cancer Res2008; 68:9280-9290).

Luminescence was recorded using a Molecular Devices Spectramax i3x platereader and data subsequently analysed using GraphPad Prism fourparameter non-linear regression model. Viability was expressed as % ofuntreated cells and calculated using the following formula:

${\%\mspace{14mu}{Viability}} = {100 \times \frac{{Luminescence}_{Sample} - {Luminescence}_{{No}\mspace{14mu}{cell}\mspace{14mu}{Control}}}{{Luminescence}_{Untreated} - {Luminescence}_{{No}\mspace{14mu}{cell}\mspace{14mu}{Control}}}}$

The % viability was plotted against the logarithm of drug concentrationin nM to extrapolate the IC₅₀ values. IC₅₀ values indicating theanti-proliferative effects of the drug conjugates are summarised inTable 4. These indicate that conjugates of the invention have improvedpotency.

TABLE 4 Cell line Drug-conjugate IC₅₀ (nM) Karpas 299 14 0.04 Karpas 29918 (comparative) 0.06 LNCaP 21 0.61 LNCaP 22 (comparative) 0.76 LNCaPMMAE (free drug) 2.14 C4-2 21 0.19 C4-2 22 (comparative) 0.27 C4-2 MMAE(free drug) 0.71

Example 10: Synthesis of Conjugation Reagent 23 (Comparative) Comprisingthe Auristatin Cytotoxic Payload, MMAE

Step 1: Synthesis of Reagent 24

A solution of 4-[2,2-bis[(p-tolylsulfonyl)-methyl]acetyl]benzoic acid(1.0 g) was added to N-hydroxybenzotriazole hydrate (306 mg) inanhydrous THF (10 mL) under a nitrogen atmosphere. The resultingsolution was cooled to 0° C. and diisopropylcarbodiimide (310 μL) wasadded dropwise. The reaction mixture was stirred for 20 min at 0° C.before being warmed to room temperature. Additional anhydrous THF (10mL) was added to the reaction mixture after 1 h. After 18 h, the formedprecipitate was filtered and washed with cold THF (2×5 mL) before beingdried in vacuo. The solid was stirred with MeOH (10 mL) for 1 h at roomtemperature, collected by filtration and washed sequentially with MeOH(2×5 mL) and Et₂O (5 mL). The solid was then dried in vacuo to givecompound 24 as a white solid (1.1 g). m/z [M+H]⁺ (618, 100%).

Step 2: Synthesis of Reagent 25

To a stirred suspension of L-Glutamic acid 5-tert-butyl ester (198 mg)in anhydrous DMF (20 mL) under a nitrogen atmosphere was added NMM (107μL). The reaction mixture was cooled to 0° C. before reagent 24 (603 mg)was added. The resulting suspension was stirred at 0° C. for 1 h, afterwhich the reaction mixture was allowed to warm to room temperature.After 19 h, the resulting solution was concentrated in vacuo andpurified by reverse phase C18-column chromatography, eluting with bufferA (v/v): water:5% acetonitrile:0.1% formic acid and buffer B (v/v):acetonitrile:0.1% formic acid (100:0 v/v to 0:100 v/v). The organicsolvent was removed in vacuo and the aqueous solvent removed bylyophilisation to give the reagent 25 as a white solid (198 mg). ¹H NMR(400 MHz, CDCl₃) δ 7.98 (1H, d), 7.86 (2H), 7.71-7.65 (6H, m), 7.36 (4H,d), 4.68 (1H, ddd), 4.34 (1H, q), 3.62 (2H, ddd), 3.50 (2H, ddd), 2.69(1H ddd), 2.55-2.45 (1H, m), 2.48 (6H, s), 2.34-2.16 (2H, m), 1.46 (9H,s); m/z [2M+H]⁺ (1371,74%), [2M+H-tBu]⁺ (1315, 70%), [M+H-tBu]⁺ (630,100%).

Step 3: Synthesis of Reagent 26

Reagent 25 (50 mg) and BOP (40 mg) were dissolved in anhydrous DMF (3mL), cooled to 0° C. and added to a solution of NH₂—PEG(24u)-OMe (99 mg)and NMM (10 μL) in anhydrous DMF (2 mL). The reaction mixture wasstirred at 0° C. and after 4 h, additional amounts of BOP (10 mg) andNMM (2.5 μL) were added to the reaction mixture which was stirred for afurther 15 min before being stored at −20° C. for 18 h. The reactionmixture was then concentrated in vacuo and purified by reverse phaseC18-column chromatography, eluting with buffer A (v/v): water:5%acetonitrile:0.1% formic acid and buffer B (v/v): acetonitrile:0.1%formic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed invacuo and the aqueous solvent removed by lyophilisation to givebis-tolylsulfonyl-propanoyl-benzamide-L-Glu-[OtBu]-[PEG(24u)-OMe] as acolourless oil (128 mg). m/z [M+H]⁺ (1757, 100%), [M+2H]²⁺ (879, 100%).Bis-tolylsulfonyl-propanoyl-benzamide-L-Glu-[OtBu]-[PEG(24u)-OMe] (126.5mg) was dissolved in formic acid (2.5 mL) and stirred under a nitrogenatmosphere at room temperature. After 20 h, the reaction mixture wasconcentrated in vacuo and dried under high vacuum for 18 h to givereagent 26 as a colourless oil (122 mg, assumed quantitative yield). m/z[M+Na]⁺ (1723, 15%), [M+H]⁺ (1700, 100%).

Step 4: Synthesis of Reagent 23

A solution of reagent 26 (13 mg), HATU (4.1 mg) and Val-Cit-PAB-MMAE.TFAsalt (9 mg) in anhydrous DMF (1 mL) under an argon atmosphere was cooledto 0° C. To this was added NMM (2 μL). After 1 h, an additional amountof HATU (4.1 mg) and NMM (2 μL) was added, and after a further 1.5 h thesolution was stored at −20° C. for 72 h. The reaction solution wasconcentrated in vacuo, dissolved in acetonitrile (1 ml) and purified byreverse phase C18-column chromatography eluting with buffer A (v/v):water:5% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v):acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). Theorganic solvent was removed in vacuo and the aqueous solvent removed bylyophilisation to give reagent 23 as a thick clear colourless oil (11.4mg). m/z [M+H]⁺ (2805, 20%), [M+2H]²⁺ (1403, 75%), [M+3H]³⁺ (936, 100%).

Example 11: Synthesis of Conjugation Reagent 27 (Comparative) Comprisingthe Auristatin Cytotoxic Payload, MMAE

Step 1: Synthesis of Compound 28

To a solution of NH₂—PEG(7u)-OMe (300 mg) in ethanol (2.5 mL) was addedtert-butyl acrylate (194 μL) and the reaction mixture was stirred atroom temperature for 72 h. The solution was concentrated in vacuo andpurified by normal phase chromatography eluting withdichloromethane:methanol (100:0 v/v to 70:30 v/v). The solvent wasremoved in vacuo to give compound 28 as a pale yellow oil (324 mg). m/z[M+H]⁺ (468, 100%).

Step 2: Synthesis of reagent 29.

To a solution of 4-[2,2-bis[(p-tolylsulfonyl)-methyl]acetyl]benzoic acid(257 mg) in anhydrous DMF (2.5 mL) at 0° C. was added HATU (204 mg) andthe reaction mixture was stirred at 0° C. for 10 min. NMM (57 μL) wasadded and the reaction solution was stirred for a further 20 min at 0°C. Additional HATU (102 mg) and NN (29 μL) were added and the solutionwas warmed to room temperature and left to stir for 30 min. After thistime, a solution of compound 28 (200 mg) in anhydrous DMF (2.5 mL) wasadded. The reaction mixture was stirred for 5 h. The solution was thenconcentrated in vacuo and purified by reverse phase C18-columnchromatography eluting with buffer A (v/v): water:55% acetonitrile:0.05%trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05%trifluoroacetic acid (100:0 v/v to 0:100 v/v). The organic solvent wasremoved in vacuo and the aqueous solvent removed by lyophilisation togive reagent 2 as a colourless solid (173 mg). m/z [M+Na]⁺ (973, 40%)[M+H]⁺ (951, 100%), [M+H-tBu]+ (895, 90%).

Step 3: Synthesis of Reagent 30

To a solution of reagent 29 (160 mg) in dichloromethane (1.6 mL) at 0°C. was added trifluoroacetic acid (0.5 mL) and the solution was stirredat 0° C. for 1 h and then stored at 4° C. for 16 h. The volatiles werethen removed in vacuo, the residue was dissolved in acetonitrile (1 mL)and diluted with aqueous buffer (v/v): water:5% acetonitrile:0.05%trifluoroacetic acid (5 mL). The solvent was then removed bylyophilisation to give reagent 30 as an amber oil (quantitative yield).m/z [M+Na]⁺ (916, 50%), [M+H]⁺ (894, 100%).

Step 4: Synthesis of Reagent 27

To a solution of reagent 30 (15 mg) in anhydrous DMF (0.6 mL) at 0° C.was added HATU (7.3 mg) and the reaction mixture was stirred at 0° C.for 20 min. NMM (2 μL) was added and the solution was stirred for afurther 20 min at 0° C. Additional HATU (7.3 mg) and NMM (2 μL) werethen added, the solution was warmed to room temperature and stirred for30 min. A separate solution of Val-Cit-PAB-MMAE.TFA salt (21.8 mg) andNN (2 μL) in anhydrous DMF (0.6 mL) which had previously been stirred atroom temperature for 10 min, was then added to the reaction solution.After stirring at room temperature for 3 h, the reaction solution wasconcentrated in vacuo and purified by reverse phase C18-columnchromatography eluting with buffer A (v/v): water:55% acetonitrile:0.05%trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05%trifluoroacetic acid (100:0 v/v to 0:100 v/v). The organic solvent wasremoved in vacuo and the aqueous solvent removed by lyophilisation togive reagent 2 as a white solid (23.7 mg). m/z [M+H]⁺ (2000, 10%),[M+2H]²⁺ (1000, 100%).

Example 12: Conjugation of Reagents 9 and 27 (Comparative) toBrentuximab to Produce Antibody Drug Conjugates (ADCs) 31 and 32(Comparative) Respectively, with DAR 4

Conjugation reagents 9 and 27 (comparative) were conjugated toBrentuximab giving rise to ADCs 31 and 32 (comparative) respectively,using a method analogous to that described in Example 3. Briefly,Brentuximab (5.0 mg/mL in 20 mM sodium phosphate, 150 mM NaCl, 20 mMEDTA, pH 7.5) was heated to 40° C. in a heating block for 15 min. TCEP(6 eq. per mAb) was added to the mAb solution, mixed gently andincubated at 40° C. for 1 h before being allowed to cool to 22° C.Conjugation reagent 9 was dissolved in acetonitrile and conjugationreagent 2 was dissolved in DMF to give 3 mM and 1.6 mM stock solutionsrespectively. The reduced mAb solutions were diluted to approximately4.3 mg/mL with 20 mM sodium phosphate, 150 mM NaCl, 20 mM EDTA, pH 7.5.Conjugation reagents (5.6 to 6.0 eq. per mAb) were added to the reducedmAb solutions to give a final antibody concentration of 4 mg/mL. Thereaction solutions were mixed gently and incubated at 22° C. for 16 to24 h. After this time, each reaction solution was treated with 50 mMN-acetyl-L-cysteine (20 eq. over reagent) at 22° C. for 1 h. Each crudeconjugation mixture was analysed by hydrophobic interactionchromatography (HIC). The crude reactions were mixed with an equalvolume of 50 mM sodium phosphate, 4 M NaCl, pH 7 and the resultingsolutions loaded onto a ToyoPearl Phenyl-650S HIC column equilibratedwith 50 mM sodium phosphate, 2 M NaCl, pH 7. The ADCs were eluted fromthe column with a gradient of 50 mM sodium phosphate, pH 7 (20%isopropanol). Fractions containing DAR 4 ADC were pooled andconcentrated (Vivaspin 20, 10 kDa PES membrane). The concentratedsamples were buffer exchanged into PBS, pH 7.1-7.5, and sterile filtered(0.22 μm PVDF membranes).

Example 13: Synthesis of Conjugation Reagent 33 Comprising a DuocarmycinCytotoxic Payload

Step 1: Synthesis of Compound 34

To a solution of Fmoc-Glu(OtBu)-OH (78 mg) in anhydrous DMF (500 μL) at0° C. was added HATU (108 mg) and NMM (34 μL) and the mixture wasstirred at 0° C. for 10 min. To this was added a solution of compound 4(44 mg) in anhydrous DMF (500 μL) and the mixture was stirred at 0° C.under an argon atmosphere for 15 min. The reaction mixture was thenconcentrated in vacuo and the residue dissolved in anhydrous DMF (500μL). Piperidine (70 μL) was added and the solution stirred for 90 min atroom temperature. The reaction solution was concentrated in vacuo andpurified by reverse phase C18-column chromatography, eluting with bufferA (v/v): water:5% acetonitrile:0.05% trifluoroacetic acid and buffer B(v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v).The organic solvent was removed in vacuo and the aqueous solvent removedby lyophilisation to give compound 34 as an orange oil (44 mg). m/z[M+H]⁺ (537, 45%).

Step 2: Synthesis of Reagent 35

To a solution of 4-[2,2-bis[(p-tolylsulfonyl)-methyl]acetyl]benzoic acid(37.5 mg) in anhydrous DMF (500 μL) at 0° C. was added HATU (65 mg) andNMM (20 μL) and the mixture was stirred at 0° C. for 10 min. To this wasadded a solution of compound 34 (44.3 mg) in anhydrous DMF (500 μL) andthe mixture was stirred at 0° C. under an argon atmosphere for 1 h. Thereaction mixture was then concentrated in vacuo, the residue dissolvedin DMF (1 mL) and purified by reverse phase C18-column chromatography,eluting with buffer A (v/v): water:0.05% trifluoroacetic acid and bufferB (v/v): acetonitrile:0.05% trifluoroacetic acid (60:40 v/v to 0:100v/v). The solvent was removed by lyophilisation to givebis-tolylsulfonyl-propanoyl-benzamide-L-Glu-(OtBu)-aza-24-crown-8 as awhite solid (28.5 mg). m/z [M+Na]⁺ (1041, 20%), [M+H]⁺ (1019, 5%). To asolution ofbis-tolylsulfonyl-propanoyl-benzamide-L-Glu-(OtBu)-aza-24-crown-8 (26.5mg) in anhydrous dichloromethane (1 mL) was added trifluoroacetic acid(500 μL) and the solution stirred at room temperature under an argonatmosphere for 1 h. The volatiles were removed in vacuo to give reagent35 as a white solid (assumed quantitative yield). m/z [M+Na]⁺ (985,35%), [M+H]⁺ (963, 30%).

Step 3: Synthesis of Compound 3

To a suspension of Boc-Val-Cit-PAB-Duocarmycin (Abzena, TCRS, 17 mg) inanhydrous dichloromethane (2 mL) at 0° C. was added trifluoroacetic acid(1 mL) and the resulting solution stirred at 0° C. for 75 min. Thevolatiles were then removed in vacuo to give compound 36 as a yellowsolid (assumed quantitative yield). m/z [M+H]⁺ (798, 100%).

Step 3: Synthesis of Reagent 33

To a solution of reagent 35 (13.4 mg) in anhydrous DMF (500 μL) at 0° C.was added HATU (13.2 mg) and NN (4 μL) and the mixture stirred at 0° C.for 10 min. To this was added a solution of compound 36 (12.7 mg) inanhydrous DMF (500 μL) and the mixture was stirred at 0° C. under anargon atmosphere for 20 min. Additional quantities of HATU (7.9 mg) andNMM (2.6 μL) were added and the reaction mixture stirred for a further80 min. The solution was then concentrated in vacuo and purified byreverse phase C18-column chromatography, eluting with buffer A (v/v):water:55% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v):acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). Thesolvent was removed by lyophilisation to give reagent 33 as a yellowsolid (5.1 mg). m/z [M+Na]⁺ (1766, 60%), [M+H]⁺ (1744, 70%), [M+Na+H]²⁺(884, 90%), [M+2H]²⁺ (872, 100%).

Example 14: Synthesis of Conjugation Reagent 37 (Comparative) Comprisinga Duocarmycin Cytotoxic Payload

To a solution of reagent 30 (14.1 mg) in anhydrous DMF (500 μL) at 0° C.was added HATU (24 mg) and NMM (7.6 μL) and the mixture stirred at 0° C.for 5 min. To this was added a solution of compound 36 (14.4 mg) inanhydrous DMF (500 μL) and the mixture stirred at 0° C. under an argonatmosphere for 40 min. The solution was then concentrated in vacuo, theresidue dissolved in water:acetonitrile (3:7 v/v, 1 mL) and purified byreverse phase C18-column chromatography eluting with buffer A (v/v):water:55% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v):acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). Thesolvent was removed by lyophilisation to give reagent 37 as a yellowsolid (8.9 mg). m/z [M+Na]⁺ (1696, 10%), [M+H]⁺ (1675, 50%), [M+Na+H]²⁺(848, 40%), [M+2H]²⁺ (838, 85%).

Example 15: Conjugation of Reagents 33 and 37 (Comparative) toBrentuximab to Produce Antibody Drug Conjugates (ADCs) 38 and 39(Comparative) Respectively, with DAR 4

Conjugation reagents 3 and 37 (comparative) were conjugated toBrentuximab, giving rise to ADCs 38 and 39 (comparative) respectively,using a method analogous to that described in Example 3. Briefly,Brentuximab (8.5 mg/mL in 20 mM sodium phosphate, 150 mM NaCl, 20 mMEDTA, pH 7.5) was heated to 40° C. in a heating block for 15 min. TCEP(6 eq. per mAb) was added to the mAb solution, mixed gently andincubated at 40° C. for 1 h before being allowed to cool to 22° C.Conjugation reagents were dissolved in propylene glycol to give 0.3 mMsolutions. Conjugation reagents (5.6 eq. per mAb) were then added to themAb solutions and the reactions mixed gently and incubated at 22° C. for42 to 78 h, during which time additional reagents (up to 1.2 eq. permAb) were added to the reactions as required. The crude reactionsolutions were then mixed with an equal volume of 50 mM sodiumphosphate, 4 M NaCl, pH 7, and 5 times the volume of the crude reactionmixture of 50 mM sodium phosphate, 2 M NaCl, pH 7. The resultingsolutions were loaded onto a ToyoPearl Phenyl-650S HIC columnequilibrated with 50 mM sodium phosphate, 2 M NaCl, pH 7. The ADCs wereeluted from the column with a gradient of 50 mM sodium phosphate, pH 7(20% isopropanol). Fractions containing DAR 4 ADC were pooled andconcentrated (Vivaspin 20, 30 kDa PES membrane), prior to being bufferexchanged into PBS, pH 7.1-7.5, and sterile filtered (0.22 μm PVDFmembranes). The samples were further purified using a Superdex 200 pgSEC column by isocratic elution with PBS, pH 7.1-7.5 (10 or 20%isopropanol). Fractions containing DAR 4 ADC were pooled andconcentrated (Vivaspin 20, 30 kDa PES membrane) prior to being bufferexchanged into PBS, pH 7.1-7.5, and sterile filtered (0.22 μm PVDFmembranes).

Example 16: Synthesis of Conjugation Reagent 40 Comprising theAuristatin Cytotoxic Payload, MMAE

Step 1: Synthesis of Compound 4

To a solution of Fmoc-Glu(OtBu)-OH (469 mg) in anhydrous DMF (8 mL) at0° C. was added HATU (419 mg) and the mixture was stirred at 0° C. for20 min. NMM (121 μL) was then added and the solution stirred at 0° C.for a further 15 min. To a separate solution of 2-aminomethyl-15-crown-5(250 mg) in anhydrous DMF (2 mL) was added NN (121 μL) and the solutionstirred at 0° C. for 20 min. The two solutions were then combined,additional quantities of HATU (419 mg) and NMM (121 μL) were added andthe mixture stirred at room temperature for 3 h. The reaction solutionwas then concentrated in vacuo and purified by reverse phase C18-columnchromatography, eluting with buffer A (v/v): water:0.05% trifluoroaceticacid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (95:5v/v to 0:100 v/v). The organic solvent was removed in vacuo and theaqueous solvent removed by lyophilisation to giveFmoc-L-Glu(OtBu)-amidomethyl-15-crown-5 as a white solid (440 mg). m/z[M+Na]⁺ (679, 25%), [M+H]⁺ (657, 30%), [M+H-tBu]⁺ (601, 100%). To asolution of Fmoc-L-Glu(OtBu)-amidomethyl-15-crown-5 (440 mg) indichloromethane (20 mL) was added piperidine (463 μL) and the solutionstirred at room temperature for 3.5 h. Additional piperidine (198 μL)was added and the mixture stirred at room temperature for a further 1.5h. The reaction mixture was then concentrated in vacuo and the residuesuspended in acetonitrile (4 mL). The acetonitrile mixture was extractedwith hexane (50 mL) before the acetonitrile layer was reduced in vacuoto give an oily residue. The residue was then purified by reverse phaseC18-column chromatography, eluting with buffer A (v/v): water:0.05%trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05%trifluoroacetic acid (95:5 v/v to 0:100 v/v). The organic solvent wasremoved in vacuo and the aqueous solvent removed by lyophilisation togive reagent 41. m/z [M+Na]⁺ (457, 10%), [M+H]⁺ (435, 100%), [M+H-tBu]⁺(379, 30%).

Step 2: Synthesis of Reagent 42

To a solution of compound 24 (82 mg) in anhydrous DMF (2 mL) was addedcompound 41 (50 mg) and NMM (12.7 μL) and the mixture stirred at roomtemperature under an argon atmosphere for 2 h. The reaction solution wasthen concentrated in vacuo, the residue dissolved in acetonitrile (500μL) and purified by reverse phase C18-column chromatography, elutingwith buffer A (v/v): water:0.05% trifluoroacetic acid and buffer B(v/v): acetonitrile:0.05% trifluoroacetic acid (70:30 v/v to 0:100 v/v).The organic solvent was removed in vacuo and the aqueous solvent removedby lyophilisation to give reagent 42 as a white solid (37.4 mg). m/z[M+Na]⁺ (939, 80%), [M+H]⁺ (917, 100%), [M+H-tBu]⁺ (861, 50%).

Step 3: Synthesis of Reagent 43

To a solution of reagent 42 (36 mg) in anhydrous dichloromethane (2 mL)was added trifluoroacetic acid (1 mL). The solution was stirred at roomtemperature for 80 min before the reaction mixture was concentrated invacuo. The residue was then dissolved in acetonitrile:water (1:2 v/v,1.5 mL) and the solvent removed by lyophilisation to give reagent 43 asa white solid (assumed quantitative yield). m/z [M+Na]⁺ (883, 75%),[M+H]⁺ (861, 100%).

Step 4: Synthesis of Reagent 40

To a solution of reagent 43 (5.6 mg) in anhydrous DMF (300 μL) at 0° C.was added HATU (2.4 mg) and the mixture stirred at 0° C. for 20 min. NN(1 μL) was added and the solution stirred at 0° C. for a further 10 min.To a separate solution of Val-Cit-PAB-MMAE.TFA salt (6.6 mg) inanhydrous DMF (300 μL) at 0° C. was added NMM (1 μL) and the solutionstirred at 0° C. for 30 min. The two solutions were then combined,additional quantities of HATU (2.4 mg) and NMM (1 μL) were added and themixture allowed to warm to room temperature under stirring for 3.25 h.The reaction solution was then concentrated in vacuo, the residuedissolved in acetonitrile:DMSO (6:1 v:v, 350 μL) and purified by reversephase C18-column chromatography, eluting with buffer A (v/v):water:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05%trifluoroacetic acid (70:30 v/v to 0:100 v/v). The organic solvent wasremoved in vacuo and the aqueous solvent removed by lyophilisation togive reagent 40 as a white solid (7 mg). m/z [M+Na]⁺ (1989, 20%), [M+H]⁺(1967, 30%), [M+2Na]²⁺ (1006, 30%), [M+H+Na]²⁺ (995, 90%), [M+2H]²⁺(984, 100%).

Example 17: Synthesis of Conjugation Reagent 44 Comprising theAuristatin Cytotoxic Payload, MMAE

Step 1: Synthesis of Compound 45

To a solution of Fmoc-Glu(OtBu)-OH (216 mg) in anhydrous DMF (8 mL) at0° C. was added HATU (193 mg) and the mixture stirred at 0° C. for 20min. NMM (56 μL) was added and the solution stirred at room temperaturefor a further 15 min. To a separate solution of compound 12 (284 mg) inanhydrous DMF (2 mL) was added NMM (56 μL) and the solution was stirredat 0° C. for 20 min. The two solutions were then combined, additionalquantities of HATU (192 mg) and NMM (56 μL) were added and the mixturestirred at room temperature for 3.5 h. The reaction solution was thenconcentrated in vacuo before the residue was dissolved in ethyl acetate(50 mL). The solution was then washed with saturated sodium hydrogencarbonate solution (2×10 mL), followed by saturated brine solution (10mL). The organic phase was then concentrated in vacuo to give crudeFmoc-L-Glu(OtBu)-aza-42-crown-14. m/z [M+Na]⁺ (1046, 20%), [M+H]⁺ (1023,100%). To a solution of crude Fmoc-L-Glu(OtBu)-aza-42-crown-14 in DMF (7mL) was added piperidine (320 μL) and the solution stirred at roomtemperature for 2 h. The reaction mixture was then concentrated in vacuoand purified by reverse phase C18-column chromatography, eluting withbuffer A (v/v): water: 0.05% trifluoroacetic acid and buffer B (v/v):acetonitrile:0.05% trifluoroacetic acid (95:5 v/v to 0:100 v/v). Theorganic solvent was removed in vacuo and the aqueous solvent removed bylyophilisation. The solid residue was then triturated with hexane (3×10mL), the solid isolated by filtration and then dried in vacuo to givecompound 45 as an off-white solid (201 mg). m/z [M+H]⁺ (801, 100%).

Step 2: Synthesis of Reagent 46

To a solution of compound 45 (86 mg) in anhydrous dichloromethane (3 mL)was added N-succinimidyl 6-maleimidohexanoate (47 mg) and the solutionstirred at room temperature. DIPEA (3×19 μL) was added to the reactionsolution after 17.5, 22.5 and 24 h. The solution was then stirred for afurther 18 h at room temperature before the reaction mixture wasconcentrated in vacuo, the residue dissolved in acetonitrile:DMSO (2:1v/v) and purified by reverse phase C18-column chromatography, elutingwith buffer A (v/v): water:0.05% trifluoroacetic acid and buffer B(v/v): acetonitrile:0.05% trifluoroacetic acid (90:10 v/v to 0:100 v/v).The organic solvent was removed in vacuo and the aqueous solvent removedby lyophilisation to give reagent 46 as a white solid (38 mg). m/z[M+Na]⁺ (1016, 30%), [M+H]⁺ (994, 20%).

Step 3: Synthesis of Reagent 47

To a solution of reagent 46 (32 mg) in anhydrous dichloromethane (1.4mL) was added trifluoroacetic acid (375 μL) and the solution stirred atroom temperature for 2.5 h. Additional trifluoroacetic acid (375 μL) wasthen added and the solution stirred at room temperature for a further 2h before the solution was concentrated in vacuo and the residue driedunder high vacuum for 18 h. The residue was then dissolved in water (1mL) and the solvent removed by lyophilisation to give reagent 47 as acolourless solid (assumed quantitative yield). m/z [M+Na]⁺ (960, 20%),[M+H]⁺ (938, 20%).

Step 4: Synthesis of Reagent 44

To a solution of reagent 47 (33.3 mg) in anhydrous DMF (750 μL) at 0° C.was added HATU (14.8 mg) and the mixture stirred at 0° C. for 20 min. NN(4.3 μL) was added and the solution stirred at 0° C. for a further 15min. To a separate solution of Val-Cit-PAB-MMAE.TFA salt (45.7 mg) inanhydrous DMF (500 μL) at 0° C. was added NMM (4.3 μL) and the solutionstirred at 0° C. for 25 min. The two solutions were then combined,additional quantities of HATU (14.8 mg) and NMM (4.3 μL) were added andthe mixture allowed to warm to room temperature under stirring for 3.5h. The solution was then concentrated in vacuo and purified by reversephase C18-column chromatography, eluting with buffer A (v/v): water:5%acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v):acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). Theorganic solvent was removed in vacuo and the aqueous solvent removed bylyophilisation to give reagent 44 as a white solid (48.3 mg). m/z[M+Na]⁺ (2066, 20%), [M+H]⁺ (2044, 25%), [M+H+Na]²⁺ (1033, 50%),[M+2H]²⁺ (1022, 100%).

Example 18: Synthesis of Conjugation Reagent 48 Comprising theAuristatin Cytotoxic Payload, MMAE

To a stirred solution of compound 13 (21.8 mg) in anhydrous DMF (500 μL)was added NN (7.8 μL). This solution was then added dropwise over aperiod of 20 min to a stirred solution of suberic acidbis(N-hydroxysuccinimide ester) (43.8 mg) in anhydrous DMF (1.5 mL) atroom temperature under an argon atmosphere. The reaction mixture wasthen stirred at room temperature for 20 h before the solution wasconcentrated in vacuo and purified by reverse phase C18-columnchromatography, eluting with buffer A (v/v): water:0.05% trifluoroaceticacid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (90:10v/v to 0:100 v/v). The organic solvent was removed in vacuo and theaqueous solvent removed by lyophilisation to give reagent 48 as a whitesolid (15.2 mg). m/z [M+Na]⁺ (2126, 10%), [M+H]⁺ (2104, 20%), [M+2Na]²⁺(1074, 50%), [M+Na+H]²⁺ (1063, 50%) [M+2H]²⁺ (1052, 100%).

Example 19: Conjugation of Reagent 44 to Brentuximab to Produce AntibodyDrug Conjugate (ADC) 49 with DAR 4.6

Conjugation reagent 44 was conjugated to Brentuximab, giving rise to ADC49, using a method analogous to that described within Example 6.Briefly, Brentuximab in 20 mM sodium phosphate buffer, 150 mM NaCl, 20mM EDTA, pH 7.5, was reduced with TCEP (2 eq. per mAb) at 40° C. for 1h. Conjugation of the reduced antibody with reagent 44 (6 eq. per mAb)was then performed. Reagent 44 was dissolved in acetonitrile to give a4.8 mM stock solution. The reduced mAb solution was diluted to 4.2 mg/mLwith 20 mM sodium phosphate buffer, 150 mM NaCl, 20 mM EDTA, pH 7.5.Reagent 44 and additional acetonitrile were added to the mAb solution togive a final antibody concentration of 4 mg/mL. The solution was mixedgently and incubated at 22° C. for 1 h. Excess reagent 44 was quenchedwith N-acetyl-L-cysteine (20 eq. over reagent) and the crude reactionmixture purified using an hydroxyapatite column equilibrated with 10 mMsodium phosphate, pH 6.7. The ADC was eluted from the column with agradient of 10 mM sodium phosphate, 2 M NaCl, pH 6.7. Fractionscontaining ADC were pooled and concentrated (Vivaspin 20, 30 kDa PESmembrane) and the concentrated sample was buffer exchanged into DPBS, pH7.1-7.5 and sterile filtered. An average DAR of 4.6 was assigned to theconjugate using the method described in Example 3.

Example 20: Conjugation of Reagent 48 to Brentuximab to Produce AntibodyDrug Conjugate (ADC) 50

Conjugation reagent 4 was conjugated to Brentuximab, giving rise to ADC50. Briefly, reagent 48 was dissolved in DMF to give a 5.3 mM stocksolution. To a Brentuximab solution (4.4 mg/mL in 20 mM sodium phosphatebuffer, 150 mM NaCl, 20 mM EDTA, pH 7.5) was added reagent 48 (10 eq.per mAb) and additional DMF to give a final antibody concentration of 4mg/mL. The solution was mixed gently and incubated at 22° C. for 1 h.The crude reaction solution was purified using an hydroxyapatite columnequilibrated with 10 mM sodium phosphate, pH 6.7. The ADC was elutedfrom the column with a gradient of 10 mM sodium phosphate, 2 M NaCl, pH6.7. Fractions containing ADC were pooled and concentrated (Vivaspin 20,30 kDa PES membrane) and the concentrated sample was buffer exchangedinto DPBS, pH 7.1-7.5 and sterile filtered. An average DAR of 4.1 forthe conjugate was calculated from the relative peak intensities of theindividual DAR species following mass spectrometry.

Example 21: Conjugation of Reagent 40 to Brentuximab to Produce AntibodyDrug Conjugate (ADC) 51 with DAR 4

Conjugation reagent 40 was conjugated to Brentuximab giving rise to ADC51 using a method analogous to that described in Example 3. Briefly,Brentuximab (5.2 mg/mL in 20 mM sodium phosphate, 150 mM NaCl, 20 mMEDTA, pH 7.5) was heated to 40° C. in a heating block for 15 min. TCEP(6 eq. per mAb) was added to the mAb solution, mixed gently andincubated at 40° C. for 1 h before being allowed to cool to 22° C.Conjugation reagent 40 was dissolved in DMF to give a 1.5 mM solution.Reagent 40 (5.6 eq. per mAb) was added to the mAb solution and thereaction was mixed gently and incubated at 22° C. for 16 h. The crudereaction mixture was mixed with an equal volume of 50 mM sodiumphosphate, 4 M NaCl, pH 7 buffer and the resulting solution loaded ontoa ToyoPearl Phenyl-650S HIC column equilibrated with 50 mM sodiumphosphate, 2 M NaCl, pH 7. The ADC was eluted from the column with agradient of 50 mM sodium phosphate, pH 7 (20% isopropanol). Fractionscontaining DAR 4 ADC were pooled and concentrated (Vivaspin 20, 30 kDaPES membrane) before the concentrated sample was buffer exchanged intoPBS, pH 7.1-7.5, and sterile filtered (0.22 μm PVDF membranes).

Example 22: Analysis of ADC 31 by In Vitro Cell Viability Assay

Brentuximab conjugate 31, containing the payload MMAE, was prepared asdescribed within Example 12. The cell viability assay using Karpas-299cells was performed as described within Example 9. Concentration rangesused are described in Table 5. IC50 values obtained are listed in Table6.

TABLE 5 Cell line Drug-conjugate Concentration range Karpas 299 31 0.4nM-0.7 pM Karpas 299 MMAE (Free Drug) 2.5 nM-1 pM  

TABLE 6 Cell line Drug-conjugate IC₅₀ (nM) Karpas 299 31 0.02 Karpas 299MMAE (Free Drug) 0.15

The IC50 value obtained for conjugate 31 shows that the ADC of theinvention has potent cell killing properties in vitro.

Example 23: Analysis of ADC 38 by In Vitro Cell Viability Assay

Brentuximab conjugate 38, containing a duocarmycin payload, was preparedas described within Example 15. The cell viability assay usingKarpas-299 cells was performed as described within Example 9. Theconcentration range used for the conjugate was 50 nM-0.6 pM and the IC50value obtained was 0.14 nM.

The IC50 value obtained for conjugate 38 shows that the ADC of theinvention has potent cell killing properties in vitro.

Example 24: Analysis of ADCs 49, 50 and 51 by In Vitro Cell ViabilityAssay

Brentuximab conjugates 49, 50 and 51, containing the payload MMAE, wereprepared as described within Examples 19, 20 and 21 respectively. Thecell viability assay using Karpas-299 cells was performed as describedwithin Example 9. Concentration ranges used for each conjugate aredescribed in Table 7. IC50 values obtained for each conjugate are listedin Table 8.

TABLE 7 Cell line Drug-conjugate Concentration range Karpas 299 49 1.0nM-0.5 pM Karpas 299 50 1.0 nM-0.5 pM Karpas 299 51 1.0 nM-0.5 pM Karpas299 MMAE (Free Drug) 2.5 nM-1 pM

TABLE 8 Cell line Drug-conjugate IC₅₀ (nM) Karpas 299 49 0.02 Karpas 29950 0.04 Karpas 299 51 0.03 Karpas 299 MMAE (Free Drug) 0.20

IC50 values obtained show that ADCs of the invention have potent cellkilling properties in vitro.

Example 25: Karpas-299 Mouse Xenograft Studies ComparingBrentuximab-Drug Conjugates 14, 31, 32 (Comparative) and Adcetris©(Comparative)

Healthy female CB17-SCID mice (CBySmn.CB17-Prkdcscid/J, Charles RiverLaboratories) with an average body weight of 18.1 g were used for cellinoculation (Day 0). 24 to 72 h prior to tumour cell injection, the micewere γ-irradiated (1.44 Gy, ⁶⁰Co). The animals were maintained in SPFhealth status according to the FELASA guidelines in housing rooms undercontrolled environmental conditions.

Tumours were induced by subcutaneous injection of 10⁷ Karpas-299 cells(T-anaplastic large cell lymphoma, ALCL) in 200 μL of RPMI 1640 into theright flank. Tumours were measured twice a week with calipers, and thevolume was estimated using the formula:

${{Tumour}\mspace{14mu}{Volume}\mspace{14mu}\left( {mm}^{3} \right)} = \frac{{width}^{2} \times {length}}{2}$

Twelve days after tumour implantation (Day 12), the animals wererandomised into groups of eight mice using Vivo manager® software (211mm³ mean tumour volume) and treatment was initiated. The animals fromthe vehicle group received a single intravenous (i.v.) injection of PBS.The treated groups were dosed with a single i.v. injection of ADC at 0.8mg/kg, or Adcetris© (brentuximab vedotin) at 1 mg/kg.

Treatment tolerability was assessed by bi-weekly body weight measurementand daily observation for clinical signs of treatment-related sideeffects. Mice were euthanized when a humane endpoint was reached (e.g.1,600 mm³ tumour volume) or after a maximum of 9 weeks post-dosing.Asterisks within the graphs indicate where animals were euthanized.

The mean tumour volumes±standard error for each treatment arerepresented in FIGS. 2a, 3a, 4a and 5a , whereas individual tumourvolumes for each treatment are represented in FIGS. 2b, 3b, 4b and 5b .All compounds were well tolerated.

Mice were dosed with 0.8 mg/kg of ADC 14, 31 or 32 or 1 mg/kg Adcetris©at day 12 post-tumour induction. Adcetris© was used at a higher dose toensure that a reduction in tumour volume could be observed for thisgroup. Each cohort dosed showed an initial reduction in tumour volume upto approximately day 20. However, after day 20, animals dosed withAdcetris© (comparative), displayed tumour re-growth in 7/8 animals, asshown by the increased average tumour volume and the individual tumourvolumes in FIGS. 2a and 2b , respectively. The graph showing the meantumour volume for this cohort (FIG. 2a ) was no longer plotted when halfof the animals had been euthanized due to tumour volume being greaterthan 1600 mm³, in accordance with accepted practice. In this group, only1/8 animals had no measureable tumour by the end of the study at day 71.

For ADC 32 (comparative), 4/8 animals had tumour re-growth after day 22post-tumour induction. This is shown in FIGS. 3a and 3b , which displayan increase in average and individual tumour volumes from day 22 to day71. Within this group, two mice were euthanized at days 41 and 68 (asindicated by an asterisk), as their tumour volumes had reached themaximum allowable size for this model. Only 4/8 animals displayed nomeasureable tumour at day 71.

For ADC 14, the response rate was markedly better than ADC 32(comparative), with 7/8 animals showing no measureable tumour volume atthe end of the study at day 71 (see FIGS. 4a and 4b ). Only one mouseshowed any tumour re-growth, which was euthanized at day 48 post-tumourinduction (as indicated by an asterisk). Even more impressively, for thecohort dosed with ADC 31, no animals showed any tumour re-growth at allafter dosing. 8/8 animals survived to the end of the study at day 71with no measureable tumour (see FIGS. 5a and 5b ).

From these studies it is clear that ADCs of the invention are moreefficacious than comparative ADCs of the prior art, with both 14 and 31displaying better tumour-killing potencies than 32 and Adcetris©.

Further Inventive Aspects

While working in this area, the inventors have found that particularlyadvantageous results can be obtained when using a particular form ofconjugation technology for conjugating a therapeutic, diagnostic andlabelling agent to a peptide or protein. Accordingly, disclosed hereinis an invention described by the following clauses.

1. A conjugate comprising a protein or peptide conjugated to atherapeutic, diagnostic or labelling agent via a linker, characterisedin that the linker includes at least two ˜(CH₂—CH₂—O—)˜ units within aring, and also includes a portion:

in which W′ represents an electron withdrawing group or a group obtainedby reduction of an electron withdrawing group, each of A and Bindependently represents a C₁₋₅alkylene or alkenylene chain, and Prrepresents said protein or peptide bonded to A and B via nucleophilesNu.

2. A conjugate as described in clause 1, which includes a portion:

3. A conjugate as described in either clause 1 or clause 2, in whicheach Nu represents a sulfur atom present in a cysteine residue in theprotein or peptide Pr; or in which each Nu represents an imidazole grouppresent in a polyhistidine tag attached to the protein or peptide Pr.

4. A conjugate as described in any one of the preceding clauses, inwhich W′ represents a —CO— or —CH(OH)— group.

5. A conjugate as described in any one of the preceding clauses, whichincludes within said ring a unit of the formula ˜(CH₂—CH₂—O—)_(x)˜ inwhich x is a number of at least 2.

6. A conjugate as described in clause 5, in which x is from 2 to 50.

7. A conjugate as described in any one of the preceding clauses, inwhich the ring is attached via a single tethering atom within the ringto the rest of the linker at a single point or at two or more points; orin which the ring is attached via two or more tethering atoms within thering to the rest of the linker at a single point or at two or morepoints.

8. A conjugate as claimed in claim 7, in which said ring has theformula:

9. A conjugate as described in clause 7, in which the ring is attachedvia two or more tethering atoms within the ring to the rest of thelinker at two or more points.

10. A conjugate as described in clause 9, in which said ring has theformula:

11. A conjugate as described in any one of the preceding clauses, whichcomprises an optionally substituted aryl or heteroaryl group immediatelyadjacent the group of formula III or IIIa; and which linker alsoincludes a —NR^(a).C(O)— or —C(O).NR^(a)— group adjacent said aryl orheteroaryl group; wherein R^(a) represents C₁₋₄ alkyl or hydrogen.

12. A conjugate as described in any one of the preceding clauses, whichincludes a therapeutic agent.

13. A conjugate as described in any one of the preceding clauses, inwhich the protein or peptide is an antibody or an antibody fragment.

14. A conjugating reagent comprising a functional group capable ofreacting with a protein or peptide, which reagent also comprises atherapeutic, diagnostic or labelling agent and a linker which includesat least two ˜(CH₂—CH₂—O—)˜ units within a ring, and in which saidfunctional group has the formula:

in which W represents an electron-withdrawing group; each of A and Bindependently represents a C₁₋₅alkylene or alkenylene chain; and eithereach L independently represents a leaving group, or both Ls togetherrepresent a leaving group; or

in which W and A have the meanings given above, L represents a leavinggroup, and m is 0 to 4.

15. A conjugating reagent as described in clause 14, in which saidfunctional group has the formula:

16. A conjugating reagent as described in either clause 14 or clause 15,in which W represents a —CO— group.

17. A conjugating reagent as described in any one of clauses 14 to 16,which includes a feature according to any one of clauses 2 to 11.

18. A conjugating reagent as described in any one of clauses 14 to 17,in which the or each leaving group includes a portion —(CH₂CH₂O)_(n)— inwhich n is a number of two or more.

19. A conjugating reagent as described in clause 18, in which the oreach leaving group has the formula —SP or —SO₂P, in which P represents agroup which includes a portion —(CH₂CH₂O)_(n)— in which n is a number oftwo or more.

20. A process for the preparation of a conjugate as described in any oneof clauses 1 to 13, which comprises reacting a protein or peptide with aconjugating reagent as described in any one of clauses 14 to 19.

21. A pharmaceutical composition which comprises a conjugate asdescribed in any one of clauses 1 to 13, in which the payload is atherapeutic agent, together with a pharmaceutically acceptable carrier,and optionally together with a further active ingredient.

DETAILED DESCRIPTION OF THIS INVENTIVE ASPECT

The reagent of the invention may be represented schematically by theformula:

in which D represents the therapeutic, diagnostic or labelling agent, Frepresents a functional grouping capable of bonding to a protein orpeptide, and

represents a ring which includes at least two ethylene glycol,˜(CH₂—CH₂—O—)˜, units. The functional grouping F is capable of reactingwith a protein or peptide as explained in more detail below.

The conjugate of the invention may be represented schematically by theformula:

in which D represents the therapeutic, diagnostic or labelling agent, F′represents the protein or peptide bonded to the remainder of theconjugate via a protein or peptide bonding portion of the linker, and

represents a ring which includes at least two ethylene glycol,˜(CH₂—CH₂—O—)˜, units.

The Polyethylene Glycol Ring

Throughout, “polyethylene glycol ring” should be understood to mean aring which includes at least two ethylene glycol, ˜(CH₂—CH₂—O—)˜, units.The ring may include two or more separate ˜(CH₂—CH₂—O—)˜ units, or itmay include one or more units of the formula ˜(CH₂—CH₂—O—)_(x)˜ in whichx is a number of at least 2. The ring may contain one or more additionalatoms to complete the cyclic structure. Additional atoms may for examplebe nitrogen, carbon, oxygen, sulfur, silicon and/or phosphorus atoms.

The ring may be attached via a single tethering atom within the ring tothe rest of the linker at a single point, or it may be attached at twoor more points. Alternatively, the ring may be attached via two or moretethering atoms within the ring to the rest of the linker at a singlepoint, or it may be attached at two or more points. Tethering atoms mayfor example be nitrogen, carbon, phosphorus or silicon atoms, especiallynitrogen and/or carbon atoms, and the atoms present at the point ofattachment to the rest of the linker may for example be nitrogen orcarbon atoms.

The following are schematic drawings of possible forms of attachment ofthe ring to the rest of the linker in conjugates or reagents of theinvention, T representing a tethering atom in the ring, and PEGrepresenting at least two ˜(CH₂—CH₂—O—)˜ units:

Specific examples of suitable rings include the following, where thesymbol ˜ indicates a point of incorporation of the ring into the linker:

Preferably the ring is attached via a single tethering atom in the ringto the rest of the linker at a single point. In another preferredembodiment, the ring is attached via two or more tethering atoms withinthe ring to the rest of the linker at two or more points.

The ring may for example consist of ˜(CH₂—CH₂—O—)_(x)˜ units in which xis at least 2, preferably from 2 to 20. Alternatively, the ring maycontain ˜(CH₂—CH₂—O—)_(x)˜ units in which x is at least 2, preferablyfrom 2 to 50, especially from 2 to 20, but may also include one or moreadditional atoms as mentioned above, or may be derivatised in some otherway.

Conjugates and reagents may be readily synthesised from crown ethers.Crown ethers are cyclic oligomers of ethylene glycol, and many differentcrown ethers are known, some of which consist entirely of ethyleneglycol units, and some of which contain additional atoms within thering. For example, aza-crown ethers contain a nitrogen atom, whilediaza-crown ethers contain two nitrogen atoms. Many crown ethers arecommercially available, and these provide convenient starting points forsynthesis of the conjugates and reagents according to the invention.Crown ethers carrying functional groups through which they may bereacted with other compounds are known, for example crown etherscarrying carboxy, hydroxy, amino, or aldehyde groups are known, as arecrown ethers fused to a benzene ring optionally carrying a functionalgroup such as a carboxy, hydroxy, amino, isocyanate, nitro or aldehydegroup.

Crown ethers are known to chelate cations, and perfluoro crown ethershave been described within U.S. Pat. No. 4,838,274 for use in MRI.Therefore the conjugates of the invention may be used in applicationswithin imaging techniques such as MRI or PET.

Typical crown ethers which can be incorporated into the conjugates andreagents according to the invention include the structures shown below.

These may be incorporated into the linker of the conjugates and reagentsof the invention by reaction through atoms, especially nitrogen atoms,present within the ring, or via groups, for example hydroxy, amino,carboxy, aldehyde, isocyanate or nitro groups, present on a side-chain.Rings having two functional groups or atoms can be attached to the restof the linker of conjugates and reagents of the invention at twoseparate attachment points, hence being incorporated into the backboneof the linker. Typical linkages are as shown below:

In addition to rings derived from crown ethers, rings derived fromcryptands may be used in the present invention. Such rings are describedfor example in US 2014/0072900, and include the following:

As an alternative to synthesising the conjugates and reagents of theinvention starting from crown ethers or cryptands, it is possible toprepare a conjugate or reagent having a PEG chain attached at one end tothe rest of the linker, and then to react the free end of the chain witha functional group present elsewhere on the linker, using conventionalchemistry. In yet another alternative synthesis method, it is possibleto prepare a conjugate or reagent containing two pendant PEG chains, andthen to create a loop by reaction of appropriate reactive groups on eachof the chains. Alkene and alkyne ring-closing metathesis may for examplebe used. All such methods of synthesis would be known to the skilledperson, and permit the preparation of a very wide range of conjugatesand reagents according to the invention, including ones in which thering is incorporated into the backbone of the linker.

The conjugates and reagents of the invention may contain one ringincluding at least two ˜(CH₂—CH₂—O—)˜ units, or they may contain two ormore such rings. The ring may be monocyclic, or it may be bi- ormulti-cyclic. Two or more rings may be attached to or incorporated intothe backbone of the linker, or they may be attached to each other, thus:

It will be understood that many different sizes and structures of ringsare possible. The important feature of the invention is that a PEG chainforms part of a cyclic structure: this chain is not a linear PEG chainwhich forms part of the backbone of the linker, neither is it a pendantPEG chain which is tethered at one end to the linker but which has afree untethered end.

In one preferred embodiment, all of the PEG in the conjugate or reagentaccording to the invention is present within one or more rings. Inanother embodiment, PEG may also be present elsewhere in the linker,specifically in the backbone of the linker or in a group linking thering to the backbone of the linker, and this is discussed in more detailbelow.

The total number of ˜(CH₂—CH₂—O—)˜ units present in the conjugates andreagents of the invention will of course depend on the intendedapplication. For some applications, high molecular weight PEGs may beused, for example the number average molecular weight may be up toaround 75,000, for example up to 50,000, 40,000 or 30,000 g/mole. Forexample, the number average molecular weight may be in the range of from500 g/mole to around 75,000. However, smaller PEG portions may bepreferred for some applications.

As with the total quantity of PEG present in the conjugates or reagentsof the invention, the number of ˜(CH₂—CH₂—O—)˜ units present in the ringwill depend on the intended application. For example the cyclic PEGportion may have a molecular weight up to 3,000 g/mole. However, cyclicgroups containing as few as 2 ethylene glycol units, for example from 2to 50 ethylene glycol units, are useful for some applications, and arepresent as a cyclic PEG group in one preferred embodiment of theinvention. PEG-containing rings with 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19 or 20 repeat units, or 24, 36, 40 or 48repeat units, may for example be used.

Conjugating Reagents and Processes

The conjugating reagent according to the invention is capable ofreacting with two nucleophiles. If two or more leaving groups arepresent, these may be the same or different. Alternatively, aconjugating reagent may contain a single group which is chemicallyequivalent to two leaving groups and which single group is capable ofreacting with two nucleophiles.

Nucleophilic groups include sulfur atoms and amine groups, andnucleophilic groups in proteins are for example provided by cysteine,lysine or histidine residues. In one preferred embodiment of theinvention, a nucleophilic group is a sulfur atom present in a cysteineresidue present in the protein. Such structures may be obtained byreduction of a disulfide bond present in the protein. In anotherembodiment, a nucleophilic group may be an imidazole group present in ahistidine residue present in a polyhistidine tag attached to theprotein.

The conjugating reagent contains the functional grouping F:

in which W represents an electron-withdrawing group, for example a ketogroup, an ester group —O—CO—, or a sulfone group —SO₂—; each of A and Bindependently represents a C₁₋₅alkylene or alkenylene chain; and eithereach L independently represents a leaving group, or both Ls togetherrepresent a leaving group. When reagents containing such groups reactwith proteins, a first leaving group L is lost to form in situ aconjugating reagent containing a functional grouping of formula:

in which m is 0 to 4, which reacts with a first nucleophile. The secondleaving group L is then lost, and reaction with a second nucleophileoccurs. As an alternative to using a reagent containing the functionalgrouping I as starting material, reagents containing the functionalgrouping II may be used, as the functional groupings I and II arechemical equivalents of each other.

These conjugating reagents of the invention are of the general typedisclosed in WO 2005/007197 and WO 2010/100430. Such reagents may forexample be used to target two sulfur atoms obtained by reduction of adisulfide bond in a protein, or imidazole groups present in histidineresidues present in a polyhistidine tag attached to a protein. It hasbeen found that the incorporation of a cyclic PEG group according to thepresent invention into reagents of this type gives particularly goodresults, with conjugation reactions occurring efficiently to producestable conjugates with a high degree of homogeneity.

A leaving group L may for example be —SP, —OP, —SO₂P, —OSO₂P, —N⁺PR²R³,halogen, or —OØ, in which P represents a hydrogen atom or an alkyl(preferably C₁₋₆alkyl), aryl (preferably phenyl), or alkyl-aryl(preferably C₁₋₆alkyl-phenyl) group, or is a group which includes aportion —(CH₂CH₂O)_(n)— in which n is a number of two or more, and eachof R² and R³ independently represents a hydrogen atom, a C₁₋₄alkylgroup, or a group P, and Ø represents a substituted aryl, especiallyphenyl, group, containing at least one substituent, for example —CN,CF₃, —NO₂, —CO₂R^(a), —COH, —CH₂OH, —COR^(a), —OR^(a), —OCOR^(a),—OCO₂R^(a), —SR^(a), —SOR^(a), —SO₂R^(a), —NHCOR^(a), —NR^(a)COR^(a),—NHCO₂R^(a), —NR^(a)CO₂R^(a), —NO, —NHOH, —NR^(a) OH, —CH═N—NR^(a)COR^(a), —N⁺R^(a) ₃, —, halogen, especially chlorine or, especially,fluorine, —C≡CR^(a), and —CH═CR^(a) ₂, in which each R^(a) independentlyrepresents a hydrogen atom or an alkyl (preferably C₁₋₆alkyl), aryl(preferably phenyl), or alkyl-aryl (preferably C₁₋₆alkyl-phenyl) group.The presence of electron withdrawing substituents is preferred.

Conjugating reagents in which P represents a group which includes aportion —(CH₂CH₂O)_(n)— in which n is a number of two or more are thesubject of our copending application GB 1418186, published as WO2016/059377, and are described above.

An especially preferred leaving group L present in a novel conjugatingreagent according to the present invention is —SP or —SO₂P, especially—SO₂P. Within this group, one preferred embodiment is where P representsa phenyl or, especially, a tolyl group. Another preferred embodiment iswhere P represents a group which includes a portion —(CH₂CH₂O)_(n)—,especially one in which n has one of the values mentioned above,especially 7. An especially preferred leaving group L is—SO₂—(CH₂CH₂O)_(n)—H/Me, especially —SO₂—(CH₂CH₂O)₇—HMe.

Throughout this Specification, any reference to a leaving group L shouldbe understood to include a specific reference to these preferred groups,especially —SO₂—(CH₂CH₂O)_(n)—H/Me, and more especially—SO₂—(CH₂CH₂O)₇—H/Me.

Preferably W represents a keto group. Preferably each of A and Brepresents —CH₂—.

Reagents of the formula I and II above form conjugates which include thegrouping F′:

in which W′ represents an electron withdrawing group or a group obtainedby reduction of an electron withdrawing group, and Pr represents aprotein or peptide bonded to A and B via nucleophiles Nu. The immediateproduct of the conjugation process (as described in more detail below)is a conjugate which contains an electron-withdrawing group W. However,the conjugation process is reversible under suitable conditions. Thismay be desirable for some applications, for example where rapid releaseof the protein is required, but for other applications, rapid release ofthe protein may be undesirable. It may therefore be desirable tostabilise the conjugates by reduction of the electron-withdrawing moietyto give a moiety which prevents release of the protein. Accordingly, theconjugation process may comprise an additional optional step of reducingthe electron withdrawing group in the conjugate. The use of aborohydride, for example sodium borohydride, sodium cyanoborohydride,potassium borohydride or sodium triacetoxyborohydride, as reducing agentis particularly preferred. Other reducing agents which may be usedinclude for example tin (II) chloride, alkoxides such as aluminiumalkoxide, and lithium aluminium hydride.

Thus, for example, a moiety W containing a keto group may be reduced toa moiety containing a CH(OH) group; an ether group CH.OR^(a) may beobtained by the reaction of a hydroxy group with an etherifying agent;an ester group CH.O.C(O)R^(a) may be obtained by the reaction of ahydroxy group with an acylating agent; an amine group CH.NH₂, CH.NHR^(a)or CH.NR^(a) ₂ may be prepared from a ketone by reductive amination; oran amide CH.NHC(O)R^(a) or CH.N(C(O)R^(a))₂ may be formed by acylationof an amine. A sulfone may be reduced to a sulfoxide, sulfide or thiolether.

Preferably the groupings F′ and F have the formula:

especially

In the above formulae, preferred leaving groups are as described above.Preferably each Nu is a sulfur atom.

Conjugating reagents according to the invention may contain more thanone functional grouping for reaction with a protein. For example, areagent may contain a functional grouping, preferably of formula I orII, at one end of the molecule, and one or more additional functionalgroupings, elsewhere in the molecule. Such structures are described infor example Belcheva et al, J. Biomater. Sci Polymer Edn. 9(3), 207-226and are useful in the synthesis of conjugates containing multipleproteins.

The novel conjugating reagents of the present invention may be preparedby methods analogous to known methods. Specific reactions areillustrated in the Examples.

Conjugating reagents according to the invention may be reacted with aprotein or peptide to form a conjugate according to the invention, andsuch a reaction forms a further aspect of the invention.

A key feature of using conjugating reagents of the formulae I or II isthat an α-methylene leaving group and a double bond are cross-conjugatedwith an electron withdrawing function that serves as a Michaelactivating moiety. If the leaving group is prone to elimination in thecross-functional reagent rather than to direct displacement and theelectron-withdrawing group is a suitable activating moiety for theMichael reaction then sequential intramolecular bis-alkylation can occurby consecutive Michael and retro Michael reactions. In reagentscontaining the functional grouping I, a leaving group serves to mask alatent conjugated double bond that is not exposed until after the firstalkylation has occurred to give a reagent including the functionalgrouping II and bis-alkylation results from sequential and interactiveMichael and retro-Michael reactions. The cross-functional alkylatingagents may contain multiple bonds conjugated to the double bond orbetween the leaving group and the electron withdrawing group.

Where bonding to the protein is via two sulfur atoms derived from adisulfide bond in the protein, the process may be carried out byreducing the disulfide bond following which the reduced product reactswith the reagent according to the invention. Preferably the disulfidebond is reduced and any excess reducing agent is removed, for example bybuffer exchange, before the conjugating reagent is introduced. Thedisulfide bond can be reduced, for example, with dithiothreitol,mercaptoethanol, or tris-carboxyethylphosphine using conventionalmethods.

Conjugation reactions may be carried out under similar conditions toknown conjugation processes, including the conditions disclosed in WO2005/007197, WO 2009/047500, WO 2014/064423, WO 2014/064424, and WO2015/057699. The process may for example be carried out in a solvent orsolvent mixture in which all reactants are soluble. For example, theprotein may be allowed to react directly with the polymer conjugatingreagent in an aqueous reaction medium. This reaction medium may also bebuffered, depending on the pH requirements of the nucleophile. Theoptimum pH for the reaction will generally be at least 4.5, typicallybetween about 5.0 and about 8.5, preferably about 6.0 to 7.5. Theoptimal reaction conditions will of course depend upon the specificreactants employed.

Reaction temperatures between 3-40° C. are generally suitable when usingan aqueous reaction medium. Reactions conducted in organic media (forexample THF, ethyl acetate, acetone, DMSO, DMF, MeCN) are typicallyconducted at temperatures up to ambient. In one preferred embodiment,the reaction is carried out in aqueous buffer which may contain aproportion of organic solvent, for example up to 20% by volume oforganic solvent, typically from 5 to 20% by volume of organic solvent.

The protein can be effectively conjugated using a stoichiometricequivalent or a slight excess of conjugating reagent. However, it isalso possible to conduct the conjugation reaction with an excessstoichiometry of conjugating reagent, and this may be desirable for someproteins. The excess reagent can easily be removed, for example by ionexchange chromatography or HPLC, during subsequent purification of theconjugate.

Of course, it is possible for more than one conjugating reagent to beconjugated to a protein, where the protein contains sufficient suitableattachment points. For example, in a protein which contains twodifferent disulfide bonds, or in a protein which contains one disulfidebond and also carries a polyhistidine tag, it is possible to conjugatetwo molecules of reagent per molecule of protein, and such conjugatesform part of the present invention.

The Payload, the Protein, the Linker, and Pharmaceutical Compositionsand Utility

All material present in the sections “The payload”, “The protein”, “TheLinker”, and “Pharmaceutical compositions and utility” above applies tothis aspect of the invention.

1. A conjugate comprising a protein or peptide conjugated to atherapeutic, diagnostic or labelling agent via a linker, characterisedin that the linker includes at least two ˜(CH₂—CH₂—O—)˜ units within aring, said ring being attached via a single tethering atom within thering to the rest of the linker, or said ring being attached via two ormore tethering atoms within the ring to the rest of the linker at asingle point.
 2. A conjugate as claimed in claim 1, which includeswithin said ring a unit of the formula ˜(CH₂—CH₂—O—)_(x)˜ in which x isa number of at least 2, or x is a number from 2 to
 50. 3. (canceled) 4.A conjugate as claimed in claim 1, in which said ring is attached via asingle tethering atom in the ring to the rest of the linker at a singlepoint.
 5. A conjugate as claimed in claim 4, in which said ring has theformula:


6. A conjugate as claimed in claim 1, which includes a therapeuticagent.
 7. A conjugate as claimed in claim 1, in which the protein orpeptide is an antibody or an antibody fragment.
 8. A conjugate asclaimed in claim 1, which includes: i) a portion:

or ii a portion:

in which W′ represents an electron withdrawing group or a group obtainedby reduction of an electron withdrawing group, each of A and Bindependently represents a C₁₋₅alkylene or alkenylene chain, and Prrepresents said protein or peptide bonded to A and B via nucleophilesNu.
 9. (canceled)
 10. A conjugate as claimed in claim 8, which comprisesan optionally substituted aryl or heteroaryl group immediately adjacentthe group of formula III or Ma; and which linker also includes a—NR^(a).C(O)— or —C(O).NR^(a)— group adjacent said aryl or heteroarylgroup; wherein R^(a) represents C₁₋₄ alkyl or hydrogen.
 11. A conjugateas claimed in claim 1, which includes a portion:˜W′—(CH═CH)_(p)—(CH₂)₂-Nu-Pr or a portion˜NH—CO—Ar—CO—(CH₂)₂-Nu-Pr in which W′ represents an electron withdrawinggroup or a group obtained by reduction of an electron withdrawing group,p is 0 or an integer from 1 to 4, and Pr represents said protein orpeptide bonded to the rest of the molecule via a nucleophile Nu. 12.(canceled)
 13. A conjugate as claimed in claim 1, in which each Nurepresents a sulfur atom present in a cysteine residue in the protein orpeptide Pr; or in which each Nu represents an imidazole group present ina polyhistidine tag attached to the protein or peptide Pr.
 14. Aconjugate as claimed in claim 1, which has the formula:

in which D represents said therapeutic, diagnostic or labelling agent,

represents said ring, and F′ represents said protein or peptide bondedto the remainder of the conjugate via a protein or peptide bondingportion of the linker.
 15. A conjugating reagent comprising a functionalgroup capable of reacting with a protein or peptide, which reagent alsocomprises a therapeutic, diagnostic or labelling agent and a linkerwhich includes at least two ˜(CH₂—CH₂—O—)˜ units within a ring, saidring being attached via a single tethering atom within the ring to therest of the linker, or said ring being attached via two or moretethering atoms within the ring to the rest of the linker at a singlepoint.
 16. A conjugating reagent as claimed in claim 15, which includesa therapeutic agent.
 17. A conjugating reagent as claimed in claim 15,in which said functional group has the formula:

in which W represents an electron-withdrawing group; each of A and Bindependently represents a C₁₋₅alkylene or alkenylene chain; and eithereach L independently represents a leaving group, or both Ls togetherrepresent a leaving group; or

in which W and A have the meanings given above, L represents a leavinggroup, and m is 0 to
 4. 18. A conjugating reagent as claimed in claim17, in which said functional group has the formula:


19. A conjugating reagent as claimed in claim 15, in which saidfunctional group has the formula:˜W—(CH═CH)_(p)—(CH₂)₂-L  (V) or˜W—(CH═CH)_(p)—CH═CH₂  (VI) in which W represents an electronwithdrawing group, p represents 0 or an integer of from 1 to 4, and Lrepresents a leaving group.
 20. A conjugating reagent as claimed inclaim 17, in which: i) the or each leaving group includes a portion—(CH₂CH₂O)_(n)— in which n is a number of two or more; or ii the or eachleaving group has the formula —SP or —SO₂P, in which P represents agroup which includes a portion —(CH₂CH₂O)_(n)— in which n is a number oftwo or more.
 21. (canceled)
 22. A conjugating reagent as claimed inclaim 15, which has the formula:

in which D represents said therapeutic, diagnostic or labelling agent,

represents said ring, and F represents said functional group capable ofreacting with a protein or peptide.
 23. A process for the preparation ofa conjugate as claimed in claim 1, which comprises reacting a protein orpeptide with a conjugating reagent, said conjugating reagent comprisinga functional group capable of reacting with a protein or peptide, whichreagent also comprises a therapeutic, diagnostic or labelling agent anda linker which includes at least two ˜(CH₂—CH₂—O—)˜ units within a ring,said ring being attached via a single tethering atom within the ring tothe rest of the linker, or said ring being attached via two or moretethering atoms within the ring to the rest of the linker at a singlepoint.
 24. A pharmaceutical composition which comprises a conjugate asclaimed in claim 1 in which the payload is a therapeutic agent, togetherwith a pharmaceutically acceptable carrier, optionally together with afurther active ingredient.